population structure, habitat study and …learningportal.wwfnepal.org/dashlib/files/population...

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POPULATION STRUCTURE, HABITAT STUDY AND MOLECULAR CHARACTERIZATION OF TAXUS BACCATA

SUBSP. WALLICHIANA (ZUCC.) PILGER: AN ENDANGERED ANTICANCEROUS PLANT OF NEPAL

By

Ms. Nistha Lamsal

Ms. Prakriti Jnawali

Ms. Sabita Kawan

Ms. Sudina Bhuju

A project report submitted in the partial fulfillment

of requirements for the degree of

Bachelor’s in Biotechnology (B. Tech)

Department of Biotechnology

School of Science

Kathmandu University

September 2013

ii



By

Ms. Nistha Lamsal

Ms. Prakriti Jnawali

Ms. Sabita Kawan

Ms. Sudina Bhuju

Under the Supervision of

Dr. Dhurva Gauchan

A project report submitted in the partial fulfillment

of requirements for the degree of

Bachelor’s in Biotechnology (B. Tech)


School of Science


September 2013

iii

ACKNOWLEDGEMENTS

We are privileged to express heartfelt gratitude to our extremely devoted supervisor Dr. Dhurva Gauchan,

Associate Professor, Department of Biotechnology, Kathmandu University, whose help, stimulating

suggestions and encouragement helped us in all time of the research and writing this thesis. He stood by

us all the way and enthusiastically guided us every simplest to sophisticated technique involved. We are

indebted by his immense support and cannot express it in words.

We are also thankful to WWF, Baluwatar for their financial support and Mr. Madhu Chhetri and entire

NTNC, MCAP team, Gorkha, for not only the financial support but also for planning the trip for us and

providing their helping hands throught the sampling procedure.

We would like to thank Dr. Tika Br. Karki, Head of Department, Department of Biotechnology,

Kathmandu University for giving us permission to do the necessary research work, use departmental

laboratory and commence this thesis. We are obliged to Dr. Bhupal G. Shrestha and Mr. Paras Timilsina

for their valuable suggestions for improvement. We are bound to Mr. Pritish Shrestha, Teaching

Assistant, Kathmandu University for his stimulating support.

We from the core of our heart would like to thank Mr. Mitra Oli for his cooperation during our work in

lab. We are thankful to all the faculty members of Department of Biotechnology, Kathmandu University

for their encouragement and co-operation during this program. We acknowledge with sense of deep

appreciation to Kathmandu University Administration and Library Department.

We owe deep respect to our family for their support, guidance, affection and critical comments. We want

to thank our dear friends for all their help, support, interest and valuable hints. We would like to express

our gratitude to all those who supported us to complete this project. Last but not the least, we would like

to appreciate every individual directly or indirectly involved in completion of our research.

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ABSTRACT



Nistha Lamsal, Prakriti Jnawali, Sabita Kawan, Sudina Bhuju


School of Science


Supervisor: Dr. Dhurva Gauchan

Associate Professor

Nepal is rich in species diversity of forest trees; physiographic and climatic variations have created

habitats for various forest tree species. However, many forest tree species are providing food and services

to the rural communities. Due to over-exploitation, important and valuable species, such as Himalayan

Yew is becoming rare and even under threat of extinction. All the merchantable size forest trees have been

logged illegally. The Government of Nepal has already banned this plant for export under the Forest Act.

This work helps to understand the status of Taxus in its natural habitat.

Himalayan yew (Taxus baccata L.) is a gymnosperm with a wide range and a discontinuous distribution.

We used RAPD markers to analyze the genetic variation among 16 yew genotypes assembled from

natural yew forests in Manaslu Conservation Area. Seven 10 mer primers were used as single primers for

amplification. A total of 132 amplified bands were obtained for the 3 primers assayed from which 100.00

polymorphism was obtained. The size range of the amplified DNAs was between 100bp to 8,000bp as

obtained by comparing with 1kb Marker DNA. Genetic variation among population was estimated using

the POPGEN32 software package, VERSION 1.31.RAPD bands separated on agarose gels were scored

and transformed into a binary matrix where presence of band was scored as 1 while absence of band was

scored as 0. Similarity and cluster analysis were conducted using Nei’s coefficient and the Unweighted

Pair Group Method with Arithmetic mean.

Key words: Yew (Taxus baccata); RAPD; Genetic diversity; Polymorphis

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CONTENTS

Acknowledgements……………………………………………………………………… iii

Abstract…………………………………………………………………………............. iv

List of Tables…………………………………………………………………………….. xii

List of Plates……………………………………………………………………………... xiii

List of Figures…………………………………………………………………………… ix

List of Illustrations……………………………………………………………………… x

Abbreviations……………………………………………………………………………. xi

Chapter I. Introduction…………………………………………………………............. 1

1.1 Plant Morphology……………………………………………………………............. 1

1.2 Taxol in Taxus……………………………………………………………………….. 1

1.3 Taxonomy……………………………………………………………………............. 2

1.4 Geographical Distribution……………………………………………………............ 2

1.5 Uses………………………………………………………………………………….. 2

1.6 Status in Nepal………………………………………………………………............. 3

1.7 Gel Electrophoresis………………………………………………………………… 3

1.8 RAPD PCR………………………………………………………………………… 4

Chapter II. Literature Review…………………………………………………………

6

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Chapter III. Population Structure and Habitat Study………………………………….. 9

3.1 Methodology………………………………………………………………………… 9

3.1.1 Vegetation Sampling and Data Analysis………………………………………... 9

3.1.2 Greenhouse Experiments……………………………………………………….. 10

3.2 Results……………………………………………………………………………….. 10

3.3 Discussions………………………………………………………………………… 12

Chapter IV. Molecular Characterization……………………………………………….. 16

4.1 Methodology………………………………………………………………………… 16

4.1.1 Plant Material………………………………………………………………….. 16

4.1.2 Isolation of Genomic DNA…………………………………………………….. 16

4.1.3 Gel Electrophoresis…………………………………………………………….. 16

4.1.4 RAPD PCR…………………………………………………………………….. 17

4.1.5 Genetic Data Analysis…………………………………………………………. 18

4.2 Results……………………………………………………………………………….. 19

4.3 Discussions………………………………………………………………………….. 27

4.3.1 Correlation between Similarity Coefficient…………………………………… 27

4.3.2 Population Genetic Analysis…………………………………………………… 29

Chapter V. Conclusion…………………………………………………………………... 31

References………………………………………………………………………………… 32

Appendices………………………………………………………………………………... 35

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LIST OF TABLES

1. Analysis of forest vegetation of the common associates of Taxus baccata subsp

wallichiana…………………………………………………………………………………...

11

2. Soil Analysis of Krak and Ongul…………………………………………………... 12

3. PCR reaction for primer OPA02…………………………………………………… 17



6. PCR Program………………………………………………………………………. 18

7. Unique PCR bands amplified using different RAPD Primers……………………... 21

8. Binary data input for POPGEN………………………………………………… 21

9. Allelic Frequency of each Locus…………………………………………………... 24

10. Summary of Genetic Variation Statistics for All Loci…………………………….. 25

11. Nei's Unbiased Measures of Genetic Identity and Genetic Distance……………… 29

12. Optimal Separation Range of Agarose…………………………………………….. 38

13. List of Primers……………………………………………………………………... 39

viii

LIST OF PLATES

1. Gel photograph of isolated genomic DNA……………………………………… 19

2. RAPD Profile generated by Primer OPA02………………………………………. 20



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LIST OF FIGURES

1. Normal Distribution Curve for Diversity Index………………………………… 28

2. Graph for Pearson’s Correlation Coefficient…………………………………… 28

3. Dendrogram Based Nei's (1978) Genetic distance: Method = UPGMA………….. 30

4. Distribution of Taxus baccata in Nepal………………………………………… 35

5. Map showing Hariyo Ban Working Area and Manaslu Conservation Area………. 35

6. Sample Collection Site…………………………………………………………… 36

7. Sample Twigs of Taxus ready for Plantation…………………………………….. 36

8. Treatment of Twig cuttings with Phytohormone…………………………………. 37

9. Result of Rooting by Mode I (Liquid Mode)……………………………………… 37

10. Result of Rooting by Mode II (Dry Mode)………………………………………. 38

x

LIST OF ILLUSTRATIONS

1. Reagents for Genomic DNA Isolation……………………………………………. 40

2. Reagents for Gel Electrophoresis…………………………………………………. 40

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ABBREVIATIONS

AFLP--Amplification fragment Length Polymorphism

CTAB--Hexadecyltrimethylammonium bromide

DNA--Deoxyribonucleic Acid

dNTP--Deoxynucleoside Triphosphate

EDTA--Ethylene-Diaminetetra-acetic Acid

MgCl2--Magnesium Chloride

HCl-- Hydrochloric Acid

ISSR--Inter Simple Sequence Repeats, A PCR based Techniques

NaOH-- Sodium Hydroxide

PCR--Polymerase Chain Reaction

POPGENE32--Population Genetics Analysis (32-bit version)

RAPD--Random Amplified Polymorphic DNA

RFLP--Restriction Fragment Length Polymorphism

RNA-- Ribonucleic acid

SSRs--Simple Sequence Repeats, A PCR based Techniques

Taq Polymerase--DNA Polymerase Enzyme isolated from Thermus aquaticus bacteria

TBE--Tris, Boric Acid and EDTA Stock Buffer

TE—Tris and EDTA Buffer

UPGMA-- Unweighted Pair Group Method with Arithmetic mean

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Chapter I

INTRODUCTION

1.1 Plant Morphology

An evergreen tree species, as most conifers, Taxus (Himalayan Yew) is a unique member of the

gymnosperms, usually 6 to 10 m in height, 1.5 to 1.8m in girth; found in temperate Himalayas at an

elevation range of 1800 to 3300m. It grows either as a small under canopy tree or a shrub. The bark is thin

reddish brown, and scaly. Leaves are dark green, linear and up to 3 cm long, with a pointed tip, and

appear to spread in two rows on either side of the shoot. The tree is dioecious in nature, having the male

and female flowers on separate trees. The female does not bear a cone but a fruit which is a solitary seed,

half enclosed in a red aril. Male flowers have globose heads on common stalk whereas the female flowers

consist of a single erect ovule surrounded by a disk, on a peduncle covered with imbricate scales. Unlike

many other conifers, Taxus does not actually bear its seeds in a cone. Instead, each seed grows alone at

the tip of a dwarf shoot, enclosed in a fleshy, usually red, aril which is open at the tip and up to 1 cm in

length. In the Himalayan region, needles are shed in May and June; flowers appear from March to May

and the fruits ripen from September to November. The tree is long lived, but the growth is extremely

slow; growth rate of 12-14 annual rings per 2.5 cm of radius and girth increment 0.4 to 1.3cm per year.

Dormancy of 1.5-2 years has been recorded (Nhut et al., 2007). When young, the seedling are demanding

in terms of moisture and shade (Svenning and Magård, 1999), but under artificial regeneration, once

established, bear transplanting capacity well. Seeds are dispersed by birds and animals, pollinated by

wind, highly clumped in distribution and not much is known about the habitat preferences the species

exhibits. Taxus is also called the 'tree of death' because the tree is poisonous however, its leaves, twigs

and barks are found to possess medicinal value.

1.2 Taxol in Taxus

The species is made more unique by the fact that it is an important medicinal plant. Wani et al. (1971)

first discovered the drug taxol which is anti-cancerous in nature. Scientists showed that the taxane

alkaloids in yew bark could be used to treat cancer, and the anti-cancer drug Taxol was approved for use

in the 1990s. As a result, there was a rush to harvest bark from Taxus brevifolia (growing in North

America) and other yew species. Chemists then found that yew leaves contained compounds that could be

used as starting material for the synthesis of Paclitaxel, the active anti-cancer compound in the drugs

Taxol and Taxotere. Eating just a few leaves can make a small child severely ill and fatalities have

occurred. All parts of the tree are poisonous, with the exception of the bright red arils. The arils are

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harmless, fleshy, cup-like structures, partially enveloping the seeds, which are eaten by birds (which

disperse the seeds); however, the black seeds inside them should not be eaten as they contain poisonous

alkaloids.

1.3 Taxonomy

Family: Taxaceae

Genus: Taxus

Species: baccata

Author: L. subsp. wallichiana(Zucc.) Pilger

Local name: Lodhsalla

English name: Himalayan Yew

The generic name Taxus is reflected in the name of the poisonous taxanes found in the tree. Some

botanists did not consider yew to be a true conifer, since it does not bear its seeds in a cone. However,

proper consideration of its evolutionary relationships now places the yew family (Taxaceae) firmly within

the conifers.

1.4 Geographical Distribution

Worldwide Taxus baccata is found in these countries:- Algeria, Australia, Belgium, Bhutan, Bosnia and

Herzegowina, Bulgaria, Canada, China, Croatia, Czech Republic, Denmark, El Salvador, Estonia,

Finland, France, Germany, Greece, Guatemala, Hungary, India, Iran, Italy, Japan, Korea, Republic of,

Lithuania, Mexico, Morocco, Nepal, Norway, Philippines, Poland, Portugal, Romania, Russian

Federation, Spain, Sweden, Switzerland, Taiwan, Province of China, Turkey, Ukraine, United Kingdom,

United States of America, Vietnam (Agroforestry Database 4.0 , Orwa et al., 2009).

In Nepal, it is distributed in the Himalayan region in the range of 1800-3000m (see appendix Figure.1) in

association with Quercus semecarpifolia, Abies spectabilis, Picea smithiana, Cedrus deodara,

Rhododendron campanulatum.

1.5 Uses

A. Subsistence Use

The foliage is used as litter and fed to cattle in Pakistan and Nepal. The red juice of the bark is used as an

inferior dye and utilized by Brahmins for staining the forehead. Bark and wood produce brown color to

the wool mordant with alum. In India, it is used for carrying poles, bows and furniture, wood of European

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species is used for turnery and fine furniture but is scarce. According to Brandis (1921) green twigs are

used to decorate the houses in Nepal during festivals.

B. Commercial Use

Extract of Taxus can be used in cosmetics, such as hair lotion, rinses, beauty and shaving cream and

dentifrices. Wood is burnt as incense. Tincture made from the young shoots has been used for the

treatment of headache, falling pulse, coldness, diarrhea. Leaves have anti spasmodic properties used in the

treatment of hysteria, epilepsy, nervousness and frequently used in Unani system of medicine.

1.6 Status in Nepal

Yearly, about 800 metric tons of crude Lodhsalla is collected by Dabur-Nepal from seven districts of

Nepal namely: Sindhuli, Dolakha, Sindhupalchowk, Manang, Gorkha, Lamjung, Ramechhap (source:

Dabur-Nepal). Himalayan yew, known for the treatment of ovarian and breast cancer has been

overexploited and smuggled heavily from eastern Nepal. Ethanol extract of its leaves is heavily exported

for manufacturing taxol for cancer treatment. Annually, in eastern region of Nepal huge quantity of fleshy

leaves and small twigs are bought at the rate of Rs. 20 per kg from the community and national forests,

and semi dried leaves and twigs normally at higher price as Rs. 50 per kg. A medium size tree normally

gives a harvest of two quintals of fleshy leaves worth providing Rs. 4,000 net without tree felling. The

leaves are pruned from September to April. Earlier, villagers used to cut down the trees for leaves but

these days they cut only the small branches. It is estimated, from a single district Sankhuwasabha, traders

export around hundred tons of semi dried leaves every year.

1.7 Gel electrophoresis

Electrophoresis is a procedure which enables the sorting, separation and sometimes purification of

macromolecules – especially proteins and nucleic acid that differ in size, charge or conformation. When

charged molecules are placed in an electric field, they migrate toward either the positive or negative pole

according to their charge. This procedure of using gel, while performing electrophoresis is called Gel

Electrophoresis. In the gel electrophoresis, the shorter molecules migrate farther than longer ones because

shorter molecules migrate more easily through the pores of the gel. This phenomenon is called sieving.

Gel Electrophoresis of large DNA or RNA is usually done by agarose gel electrophoresis. Agarose is a

natural polysaccharide, purified from seaweed. Agarose gel has large range of separation but relatively

low resolving power. By varying the concentration of agarose fragments of DNA from about 200 to

50,000 base pair can be separated using standard electrophoresis technique (see appendix Table.1).

Agarose gels are easy to cast and handle compared to other matrices and nucleic acids are not chemically

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altered during electrophoresis. The distance between DNA bands of a given length is determined by

percent of agarose in gel.

The following factors determine the rate of migration of DNA through agarose gels.

The molecular size of the DNA

Molecules of double standard DNA migrate through gel matrices at rates that are inversely

proportional to the log10 of the number of base pairs. Larger molecules migrate slowly because of

greater frictional drag and they worm their way through the pores of gel less efficiently than smaller

molecules. So, they remain closer to the wall.

The concentration of agarose

Most agarose gels are made with between 0.7% (good separation or resolution of larger 5-10kb DNA

fragments) and 2% (good resolution for small 0.2-1kb fragments) agarose dissolved in electrophoresis

buffer.

The applied voltage

As voltage applied to gel increases, larger fragments migrate faster than smaller ones. Hence, the best

resolution of fragments larger than 2kb is attained by applying no more than 5-8 volts per cm to the

gel.

The electrophoresis buffer

The electrophoretic mobility of DNA is affected by the composition and ionic strength of the buffer.

The most commonly used for duplex DNA are TAE( Tris-acetate-EDTA) and TBE ( Tris-borate-

EDTA). It not only establishes pH but provides ions to support conductivity. Use of water as buffer

will result no migration of DNA in the gel.

Effects of Ethidium Bromide (fluorescent dye that binds in between DNA)

Interaction of ethidium bromide causes decrease in negative charge and an increase in both stiffness

and length of double stranded DNA.

1.8 RAPD PCR ( Random Amplified Polymorphic DNA, Polymerase Chain Reaction)

The RAPD procedure was introduced by William et al (1990). It employs single primer with 10

nucleotides and a GC content of at least 50%. RAPD marker is classified as dominant marker. The

brightness of the band depend on several factors including the degree of repetitiveness of the targeted

DNA region, the extent of primer-template mismatch, and the presence or absence of competing target

regions in the genome.

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RAPD polymorphisms can theoretically result from several types of events:

a. Insertion of a large piece of DNA between the primer binding sites may exceed the capacity of

PCR, resulting in fragment loss.

b. Insertion or deletion of a small piece of DNA will lead to a change in size of the amplified

fragment

c. The deletion of one of the two primer annealing sites results in either the loss of a fragment or an

increase in size

d. A nucleotide substitution within one or both primer target sites may affect the annealing process

which can lead to presence versus absence polymorphism or to change in fragment size.

RAPD has its advantages over its simplicity, requirement for low quantities of template DNA, no prior

requirement of sequence for primer, easy and quick to assay with low costs involved. Disadvantage that

RAPD share with other marker is their dominant nature, which limits their use for population genetics and

mapping studies. RAPD is sensitive to slight changes in the reaction conditions and results in low

reproducibility. Main application areas includes the identification of cultivars and clones, genetic

mapping, marker-assisted selection, population genetics, and molecular systematic at the species level.

Polymerase chain reaction (PCR) is the technique of in-vitro amplification of DNA. Amplification is

usually carried out by DNA polymerase I enzyme from Thermus aquaticus. This species live in hot

springs and enzymes produced by them are thermostable. The thermostable DNA polymerase adds

nucleotide in 3' end of a primer. To begin, the enzyme is added to complementary strands. The mixture is

heated to 94 C so that newly synthesized strands detach from the template. Then, it is cooled enabling

more primers to hybridize at their respective positions. Taq polymerase unlike most types of DNA

polymerase is not inactivated by the heat treatment and carries out a second round of DNA synthesized.

The cycle of denaturation – hybridization synthesis is repeated usually 25-30 times, resulting synthesis of

several hundred million copies of the amplified fragment. If sufficient DNA has been produced from the

amplified fragment, it is visible as discrete band after staining with EtBr while performing gel

electrophoresis. This may provide useful information about the DNA region that has been amplified.

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Chapter II

LITERATURE REVIEW

Gymnosperms are ancient plant groups having their origin in the Permian era, 200-300 million years ago

(Beck, 1966). They exhibit extremely interesting palaeo history by the fact that they have a comparatively

intact fossil record and are represented by relatively few extant taxa i.e., 63 genera and 750 species

(Sahni, 1990). Though outnumbered by angiosperms in terms of species richness, gymnosperms,

especially the conifers dominate the world’s forest types (GBO, 2001), occurring across huge landscapes

of the temperate zones of both hemispheres. However, many species are faced with extinction as single

populations stand as the last living examples (Sahni, 1990). A unique family among the gymnosperms,

whose position in the classification systems has been a source of controversy, is Taxaceae. Earlier

belonging to the order Coniferales (cone bearing plants), the family was shifted out to a separate order of

itself called Taxales (Sahni, 1990). The justification being, that Taxaceae did not bear cones. In India, the

family is represented by two genera, namely Taxus and Amento Taxus (Sahni, 1990).

Taxus is a unique member of the Gymnosperms. It is dioecious in nature, dispersed by birds and animals,

pollinated by wind, highly clumped in distribution and not much is known about the habitat preferences

the species exhibits. The species is made more unique by the fact that it is an important medicinal plant.

Wani et al. (1971) first discovered the drug taxol which is anti-cancerous in nature. Without any

knowledge of its status in the wild and scanty knowledge about its ecological requirements, the

injudicious use of the plant for commercial purposes is enough to drive the species towards its extinction.

Populations may be vulnerable and factors such as population size, degree of isolation and fitness

(Ellstrand and Elam, 1993) can help predict their fate. Not yet known to be successfully regenerated by

artificial means, and characterized by poor regeneration in nature (Svenning and Magård, 1999), an

insight into its microhabitat preferences will be useful for future assisted regeneration programmes.

Taxol (Paclitaxel) is an important antitumor agent. It was first isolated from the banks of the Pacific yew

(Taxus brevifolia Nutt.) in 1971 (Wani et al., 1971) and later found in other Taxus species, including

Taxus wallichiana var. mairei (Lemee & Leveille) L. K. Fu & Nan Li (Ho et al. 1997). Because it can kill

tumor cells by enhancing the assembly of microtubules and inhibiting their depolymerization, taxol has

been well established and approved by FDA (Food and Drug Administration) as a very important

effective chemotherapeutic agent against a wide range of tumors since 1992. Subsequently, attempts were

undertaken to synthesize taxol, but the supply of taxol and is precursors continues to rely on biological

sources (Suffness, 1995).

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Taxus wallichiana var. mairei is the most widespread variety of T. wallichiana (Fu et al., 1999; Moller et

al., 1999; Moller et al., 2007) and is mainly distributed in the mountain regions of East and South China

and on the islands of Taiwan. Before T. wallichiana var. mairei was listed as first-class conservation plant

in 1995 (Yu, 1995), it suffered twofold- from overexploitation and from habitat loss. The variety’s

remaining populations are greatly disjunct and restricted to a few protected areas and very remote

localities. Taxus wallichiana was listed as endangered in the China plant Red Book (Fu, 1992), although

this listing relates only to var. wallichiana and not to the species as whole. However, in 2004 all varieties

of Taxus wallichiana and other closely related Asian yews were added to Appendix 2 of the Convention

on International Trade in Endangered Species to protect them from illegal trade (TRAFFIC, 2004).

Population studies on yew reveal interesting patterns and life history strategies. Shrubs have been

reported to assist the establishment of Yew woodlands (Svenning and Magård, 1999). But once the

woodlands oust the shrubs, regeneration of Taxus is hampered. Taxus also inhibits self-regeneration and

seedling survival, prompting concerns for the long-term preservation of the species. Pre germination,

factors that influence establishment are availability of mother trees (seed source), availability of perch

sites, availability of dispersers and availability of suitable habitat for germination, presence of seed

predators (García et al., 2005). Since, Taxus falls under k- strategist species, it is density dependent and

availability of suitable habitat plays an important role. Post germination factors like optimum light,

nutrient requirement, protection from grazing are also key to the survival of the species.

Few studies on genetic variation in T. baccata are available to date. These studies, based on the analyses

of isozymes, concern either only a single stand (Lewandowski et al. 1995; Rajewskiet al., 2000) or few

populations and ornamental trees to determine genetic variation (Hertel & Kohlstock, 1996). More recent

studies in Taxus Canadensis and Taxus brevifolia have used isozymes, RAPDs, restriction fragment

length polymorphisms (RFLPs), or amplified fragment length polymorphisms (AFLPs) to elucidate clonal

variation within populations, population dynamics, or metapopulation structure (El-Kassaby & Yanchuk,

1994; Wheeler et al., 1995; Scher, 1996; Senneville et al., 2001; Corradini et al., 2002). RAPD marker

was also used for regional differentiation in Swiss populations of English yew (Hilfiker et al., 2004) and

to assess the genomic diversity of individual plants within a T. cuspidata population (Li et al., 2006).

Extraction of a suitable quantity and quality of DNA is important for any molecular analysis. The PCR

depends on the uses of a specific class of enzymes, DNA polymerase. PCR can amplify a single copy of

template DNA exponentially to yield sufficient DNA for analysis. PCR can be performed by arbitrarily

designed primers (Williams et al., 1990; Welsh and MeClellend, 1990) and by specifically designed

primers (Weising et al, 1994). Over the last decade, polymerase chain reaction (PCR) technology has

8

become a widespread research technique and has led to the development of several novel genetic assays

based on selective amplification of DNA.

RAPD markers are a modification of PCR contrived in the late 1980’s (Wiliams et al., 1990). PCR is

becoming more powerful with the introduction of user-friendly and fully automated techniques (Kreader

et al., 2001). The random amplified polymorphic DNA (RAPD) technique can reveal polymorphism

between very closely related genotypes. Since 1990, RAPD markers have been successfully used to

identify cultivars and clones of barley (Tinker et al., 1993), wheat (He et al.,1992), broccoli, cauliflower

(Hu & Quiros, 1991; Demeke et al., 1992), apple (Koller et al., 1993; Autio et al., 1998; Oraguzie et al.,

2001), pear (Monte-Corvo et al., 2000), almond (Bartolozzi et al., 1998), apricot (Takeda et al.,1998),

olive (Claros et al., 2001; Besnard et al., 2001; Belaj et al., 2001), pistachio (Hormaza et al., 1998),

walnut (Nicese et al., 1998) and bluegrass (Rajasekar et al., 2006).

The primary drawback to RAPD markers is that they are dominant and do not permit the scoring of

heterozygous individuals and low reproducibility. Despite above drawbacks, the RAPD method will

probably be important as long as other DNA-based techniques remain unavailable in terms of cost, time

and labor. Perhaps the main reason for the success of RAPD the gain of a large number of genetic

markersthat require small amounts of DNA without the requirement for cloning, sequencing or any other

form of the molecular characterization of the genome of the species. RAPD markers have found a wide

range of applications in gene mapping, population genetics, molecular evolutionary genetics and plant

and animal breeding. This is mainly due to the speed, cost and efficiency of the RAPD technique to

generate large numbers of markers in a short period compared with previous methods (Bardakci, 2001).

9

Chapter III

POPULATION STRUCTURE AND HABITAT STUDY

3.1 Methodology

The study was conducted in the Manaslu Conservation Area (MCA) ranging from 2000 to 3900 m.

Established in 1998, MCA covers an area of 1,663 km2 of 7 VDCs in the Mansiri Himal range in Gorkha

District. It is a classic setting of pristine mountain nature and culture. The conservation area starts at

600m and reaches to Mt. Manaslu at 8,613m, the eighth highest peak in the world. MCA is mainly

drained by Budhigandaki and Daraudi rivers. MCA falls within the boundary of Chitwan-Annapurna

Landscape (CHAL), one of Hariyo Ban Program’s priority working areas (see appendix Figure.2).

3.1.1 Vegetation Sampling and Data Analysis:-

By interacting with the local people and staffs of MCA Office, Philim, it was found that till now Taxus

has been seen and reported only in two VDCs within MCA’s territory i.e., Bihi VDC and Prok VDC

among which the Bihi VDC was more accessible site. Hence, Bihi VDC was fixed as the working site

(see appendix Figure.3). With the help of local people of Bihi VDC, two villages Krak (2455m) and

Ongul (2550m) of Bihi was found to have Taxus trees. Hence these two villages became the sampling

sites.

For vegetation sampling in each site, 50 x 50m plot, five 10 x10m quadrats was laid randomly for

investigation of species richness and other vegetation parameters. Trees and shrubs were recorded in 10 x

10m. Each 10 x 10 m quadrat was subdivided into 2 x 2m sub-quadrat for seedlings and saplings and 1 x

1m for herbs (Misra, 1968; Muller-Dombois and Ellenberg, 1974;Curtis, 1959). All the plants within this

plot were observed carefully and all the needed data were collected required for the analysis.

For DNA extraction, leaves, twigs and barks of Taxus baccata were collected from the two sites, each tree

being 50m apart from each other. Besides, to carry out soil analysis, soil sample were also collected from

each site. Soil samples were collected of ground level, 10cm below ground level and 20cm below ground

level respectively. All the samples were tagged and wrapped in the zipper bags properly and refrigerated

at -20°C when in use. Soil samples were sent to instrumental lab to analyze pH, moisture and

conductivity.

http://en.wikipedia.org/wiki/Mansiri_Himal

http://en.wikipedia.org/wiki/Gorkha_District

http://en.wikipedia.org/wiki/Gorkha_District

10

3.1.2 Greenhouse Experiments:-

1. Vegetative propagation

The twigs of Taxus were taken brought from the sampling site. 6-8inches long cuttings were made out of

the twigs such that each possessed node regions (see appendix Figure.4). The lower portion of the twigs

were cleaned and treated with phyto-hormones and then planted on the prepared soil bed in the

greenhouse. Cuttings were watered regularly depending upon the weather conditions and soil moisture

status. The cuttings were protected from direct sunlight and to maintain high humidity (70%) by a thatch

covering. The cuttings were observed periodically for initiation of rooting until rooting is completed.

2. Treatments of cuttings:

The cuttings were treated with the phyto-hormone Indole Butyric Acid (IBA)at different concentrations

(5,000 and 10,000 PPM) and two different modes of applications for induction of rooting. The mode -I

consisted of dipping the cuttings in aqueous hormonal solution(s) for 18 hours prior to planting, while the

mode-2 comprised of application of dry hormone(s) mixed thoroughly with talcum powder immediately

at the time of planting (see appendix Figure.5).

3.2 Results

After three months growth of Tap Root System from the planted Taxus twigs were seen. Mode I, where

twigs were dipped in aqueous solutions of phyto-hormone gave no any root system (see appendix Figure

6) whereas Mode II, where twigs were dipped in dry powder of phyto-hormone gave great results (see

appendix Figure 7). Hence, dry mode is better for Taxus transplantation and propagation.

Quadrat data were pooled by plots to estimate density, frequency (%), frequency class, abundance, and

relative values of density, dominance and frequency (Misra, 1968; Muller-Dombois and Ellenberg, 1974).

Importance value index (IVI) was calculated by summing up the relative values of density, frequency, and

dominance. i.e.,

IVI = Relative Density + Relative Frequency + Relative Dominance

Where,

Relative Density = (Total Individuals, Species A) / (Total Individuals, All Species)

Relative Frequency = (Frequency Value, Species A) / (Total Frequency Values, All Species)

[Frequency value = (Intervals, Points where species A occurs) / (Total Sample Points)]

Relative Dominance = (Basal Area or Coverage, Species A) / (Total Basal Area or Coverage, All Species)

11

Table 3.2.1 Analysis of forest vegetation of the common associates of Taxus baccata subsp

wallichiana

12

In case of soil analysis, three parameters were studied; moisture, pH and conductivity. Moisture

percentage was obtained by keeping the soil in the oven at 85 C for 48hours. pH and conductivity of

each soil samples were obtained by using electrochemical analyzer (Consort C6030, Consort bvba,

Belgium).

Table 3.2.2 Soil Analysis of Krak and Ongul

Sample pH Moisture Conductivity(µS/m) Krak 0 7.22 53.79% 217 Krak 10 7.41 78.05% 186.8 Krak 20 7.5 86.06% 296 Ongul 0 7.24 62.29% 85 Ongul 10 7.57 72.22% 62.1 Ongul 20 7.79 75.64% 258

0 means ground level, 10 means 10 cm below ground level and 20 means 20 cm below ground level.

3.3 Discussion

In a general sort of way, the nature of communities is controlled by either physical or abiotic conditions

such as substrate, the lack of moisture, and wave action or by some biological mechanism. Biologically

controlled communities are often influenced by a single species or by a group of species that modify the

environment, these organisms are called dominants. According to Table 3.2.1 Pinus wallichiana has the

highest value for relative dominance i.e., 9.33 and then Cornus capitatus with relative dominance value

5.61 compared to Taxus baccata subsp wallichiana with relative dominance value 4.69. Hence, these

plants can be considered to be dominant plant in that community. But it is not easy to describe a dominant

or to determine the dominant species. The dominant in a community may be the most numerous, possess

the highest biomass, preempt the most space, make the largest contribution to energy flow or mineral

cycling, or by some other means control of influence the rest of the community. In a practical sense,

according to some ecologists, in a forest the small understory trees can be numerically dominant, yet the

nature of the community is controlled by a few large trees that over-shadow the smaller ones. Hence,

overall the IVI value i.e., the Importance Value Index is more important than the dominance value as it

not only accounts for relative dominance but also relative frequency and relative density value and also as

this value determines within the community which plant is more important and has the prior advantage of

preservation. From the Table. 3.2.1 it is clear that Taxus baccata subsp wallichiana has the highest IVI

value hence it is the most important plant in that community and must be kept in priority for preservation.

Thus, Pinus wallichiana may be the dominant plant but Taxus can be the controlling plant of this

community.

13

The concept of dominance involves other implications too like; dominant species achieve their status by

occupying niche space that might potentially be occupied by other species in the community. For

example, Euphorbia wallichii has lowest importance value. This indicates it has a very low role in

ecosystem and its existence is questionable. Hence, when this plant will extinct the space occupied by this

plant can be filled by Taxus uplifting its importance value as well as dominant feature. However, a

species or small group of species to achieve dominance, it must relate to a total population of species, all

of which have similar ecological requirements; it must be able to exploit the range of environmental

requirements more efficiently than other species in same trophic level) Hence, Taxus baccata must have

obtained its importance value in this community because of accomplishments of all above mentioned

criteria making its importance value 22.92, highest of all.

From the table, it also clears that Taxus forest is rich in many different plants like Cynodon dactylon,

Berberi asiatica, Cornus capitatus, Pinus wallichiana, Arisaema tortuasum, Quercus semicapifolia,

Thalicitrum spp, Mahonia napulensis, Asparagus nepalensis etc., making it an ensemble community in

itself.

For the study of soil three parameters are very important; pH, moisture percentage, and conductivity. The

acidity or alkalinity of soil is expressed on a pH scale, based on the total hydrogen ion concentration in

the soil water solution. The soil sample that we had collected from Krak and Ongul are found to be

slightly alkaline in nature i.e., pH ranging from 7.2 – 7.8 which may be due to an accumulation of soluble

salts of Na+, K+, Mg++, Ca++. The pH of soil increased as we went deeper inside the ground.

Usually plants must maintain a neutral charge in their roots. In order to compensate for the extra positive

charge, they release H+ ions from the root. Excess rainfall leaches base cation from the soil, increasing the

percentage of Al3+ and H+ relative to other cations. Additionally, rainwater has a slightly acidic pH of 5.7

due to a reaction with CO2 in the atmosphere that forms carbonic acid. Ammonium (NH4+) fertilizers

react in the soil in a process called nitrification to form nitrate (NO3 ), and in the process release H+ ions.

Some plants will also exude organic acids into the soil to acidify the zone around their roots to help

solubilize metal nutrients that are insoluble at neutral pH, such as iron (Fe). Both primary and secondary

minerals that compose soil contain Al. As these minerals weather, some components such as Mg, Ca, and

K, are taken up by plants, others such as Si are leached from the soil, but due to chemical properties, Fe

and Al remain in the soil profile. When atmospheric water reacts with sulfur and nitrogen compounds that

result from industrial processes, the result can be the formation of sulfuric and nitric acid in rainwater.

Decomposition of organic matter by microorganisms releases CO2 which when mixed with soil water

forms weak carbonic acid (H2CO3). Hence the processes contributing to the formation of acid soils are

14

rainfall, fertilizer use, plant root activity and the weathering of primary and secondary soil minerals. Plant

vary in their tolerance to soil acidity. Acidic soil often causes the stunting and yellowing of leaves,

resulting in the decrease in growth and yield of crops as the pH level falls. Below pH 4, H+ ions

themselves damage root cell membranes.

Basic soils have a high saturation of base cations (K+, Ca2+, Mg2+ and Na+). This is due to an

accumulation of soluble salts which are classified as either saline soil, sodic soil, saline sodic soil or

alkaline soil. All saline and sodic soils have high salt concentrations with saline soils being dominated by

calcium and magnesium salts and sodic soils being dominated by sodium. Alkaline soils are characterized

by the presence of carbonates. Soil in areas with lime stone near the surface are alkaline from the calcium

carbonates in limestone constantly mixing with the soil. Groundwater sources in these areas contain

dissolved limestone.

The availability of plant nutrient is considerably affected by soil pH. Calcium, magnesium, sodium are the

alkaline elements, which are lost with increasing acidity whereas phosphorus is more available in acidic

soil condition. Acidity can also induce deficiencies of micronutrients such as molybdenum, copper and

boron, although deficiency in the latter is more commonly seen in alkaline soil where over liming has

occurred. Plants grown in acid soils can experience a variety of symptoms including aluminium (Al),

hydrogen (H+), and/or manganese (Mn) toxicity. Ammonium toxicity is the most widespread problem in

acid soils. Aluminium is present in all soils, but dissolved Al3+ is toxic to plants; Al3+ is most soluble at

low pH, above pH 5.2 little aluminum is in soluble form in most soils. Aluminium is not a plant nutrient,

and as such, is not actively taken up by the plants, but enters plant roots passively through osmosis.

Aluminium damages roots in several ways: In root tips and Aluminium interferes with the uptake of

Calcium, an essential nutrient, as well as bind with phosphate and interfere with production of ATP

and DNA, both of which contain phosphate. Aluminium can also restrict cell wall expansion causing

roots to become stunted.

In soils with high content of manganese (Mn) containing minerals, Manganese toxicity can become a

problem at pH 5.6 and below. Manganese, like aluminium becomes increasingly more soluble as pH

drops, and Manganese toxicity symptoms can be seen at pH's below 5.6. Mn is an essential plant nutrient,

so plants transport manganese into leaves. Classic symptoms of manganese toxicity are crinkling or

cupping of leaves.

Hence, because of the basic pH of the soil of Krak and Ongul growth of Taxus has been favored there as

alkaline pH helps in not only storage of plant nutrient like calcium, magnesium and sodium but also

prevents from suffering from toxicities.

15

Moisture percentage indicates amount of water contained in the soil. According to Table 3.2.2, the

moisture content of soil is increasing as we go deeper inside the ground level. This indicates water

collected from rainfall and humid air has been stored in the pores of soil that helps the plants especially

the roots to grow properly.

In the soil, the electric conductivity reading shows the level of ability the soil water has to carry an

electric current. The electric conductivity level of the soil is a good indication of the amount of nutrient

available for the plant to absorb. All the major and minor nutrients important for plant growth take the

form either cations (+vely charged ions) or anions (-vely charged ions). These ions that are dissolved in

the soil water carry electric charge and thus determine electric conductivity level of the soil and how

many nutrients are available for the plant to take in. the soil conductivity of Krak and Ongul is ranging

from 62-296µS/m according to Table 3.2.2. This large difference in soil conductivity indicates variability

in the soil profile and soil permeability making them favorable for plant growth. Soil conductivity is also

related to specific soil properties such as topsoil depth, pH, salt concentration, and water holding capacity.

16

Chapter IV

MOLECULAR CHARACTERIZATION

4.1 Methodology

4.1.1 Plant Material

A total of 16 samples of Taxus baccata were collected 8 each from two villages of Bihi VDC, Krak and

Ongul that lies inside the Manaslu Conservation Area, Gorkha. The samples were tagged as K1-K8 for

samples from Krak and O1-O8 for samples from Ongul. The samples were wrapped in jipper bags and

refrigerated at -20°C when not subjected to immediate use.

4.1.2 Isolation of Genomic DNA

Leaves of Taxus baccata were harvested and were pulverized using Liquid Nitrogen with help of mortar

and pestle (mid-ribs of the leaves were removed). Pulverized samples were transferred into 1.5ml

eppendorf tubes up to the 0.5 marked level. 0.7ml (700µl) Lysing Buffer was added in each tubes.

Samples were incubated at 55-60 C for about an hour. Samples were then centrifuged at 9,500×g for

5mins. Aqueous phase was carefully transferred to a fresh tube and equal volume of Chloroform-Isoamyl

alcohol was added in each tubes. Samples were again centrifuged at 9,500×g for 5mins. The supernatant

was transferred to a new tube. 0.2ml (200µl) 5M Sodium Acetate was added in this stage in order to have

excess quality of extracted DNA. Equal volume of Isopropanol was added in each tubes. Samples were

kept in -20 C for 30mins. Samples were centrifuged at 11,500×g for 5mins. The supernatant was removed

and 500µl of 96% Ethanol was added in each tubes. Samples were centrifuged at 7,000×g for 5mins. The

supernatant was removed and 500µl of 70% Ethanol was added in each tubes. Samples were centrifuged

at 7,000×g for 5mins, to stick pellets at the bottom of the tube. The supernatant was discarded and tubes

were allowed to dry at room temperature. A volume of 30-50µl TE Buffer was added to dissolve the

pellet and the DNA solution was stored at -20 C.

4.1.3 Gel Electrophoresis

1. Preparation of the Agarose Gel

0.8% agarose was weighed and transfered in conical flask containing 30ml of 0.5X TBE. Agarose

solution was completely melted by heating in microwave oven in medium temperature for 2 minutes.

3µl of Ethidium bromide was added to the warm agarose and gently mixed. Then slowly the warm

agarose gel was poured in a casting tray (sealed both ends with tape) with a comb and left for about

17

15 minutes at room temperature for gel formation. The tape and comb was removed slowly from the

tray when the gel becomes hard. The tray was placed on the tank filled with 0.5X TBE such that the

gel was completely submerged in TBE buffer.

2. Loading the sample DNA

5µl of sample DNA was mixed with 3µl of gel loading buffer. The mixture was loaded in the well

using micro pipettes. The electrophoresis was carried out at 90volt for 30mins. The bubble formation

in the buffer was checked to confirm electrophoresis. Once the first tracking dye migrated about 2/3

of the gel, the process was stopped. The gel was then placed under UV in the gel documentation

system to observe the result. The gel was photographed using CANON Powershot camera for further

analysis.

4.1.4 RAPD PCR ( Random Amplified Polymorphic DNA, Polymerase Chain Reaction)

Table 4.1.4.1 PCR Reaction for Primer OPA02

S.N. Ingredients µl for a 25µl Reaction

1. Sterile Water 10.2

2. DNA 2.0

3. 10mM Primer 2.0

4. Master Mix 10.0

5. 25mM MgCl2 0.5

6. Tag Polymerase 0.3




2. DNA 2.0

3. 10mM Primer 2.0

4. Master Mix 10.0

5. 25mM MgCl2 2.5



18



2. DNA 2.0

3. 10mM Primer 2.0

4. Master Mix 10.0

5. 25mM MgCl2 2.5


Table 4.1.4.4 PCR Program

Steps Temperature Time

1. Initiation Denaturation 94 C 3 mins

2. Denaturation 94 C 15 secs

3. Annealing 35 C 30 secs

4. Extension 72 C 1 min. 30 secs

5. Repeats Repeat step 2-4, 44 times -

6. Final Extension 72 C 3 mins

7. Hold 4 C Hold

End

The result was then observed by carrying out gel electrophoresis.

4.1.5 Genetic Data Analysis

Polymorphic bands were scored as binary data (present = 1; absent = 0) across the genotypes to generate a

binary data matrix. The matrix was then used to generate a Genetic similarity (GS) matrix. Genetic

variation was estimated using the POPGEN 32 software package, VERSION 1.31 ( Yeh, Yang and Boyle

1999) and the following indices were calculated: number of alleles per locus, effective number of alleles

per locus, percentage of polymorphic loci, Nei’s (1973) gene diversity (h*) and Shannon’s index (I*). The

latter was adopted assuming the populations to be in Hardy-Weinberg equilibrium, an outcrossing mating

system, and nearly random mating. Nei's Unbiased Measures of Genetic distance was used to construct a

dendrogram using UPGMA algorithm.

4.2 Results

19

The sizes of the isolated genomic DNA ranged from10,000bp to above (Plate 4.2.1). Out of 7 arbitrary

primers screened, 3 revealed clear and distinct polymorphisms in 16 varieties producing a total of 29

polymorphic loci. The percentage of polymorphism of the amplified DNA fragments was calculated as

100.00. Primer OPA02, OPA10 and OPA18 were able to distinguish all the Taxus genotypes. The GC%

of all the primers was above 50% (Table.4.2.1). The banding pattern of the genotypes using these

respective primers are shown in Plate 4.2.2, 4.2.3 and 4.2.4. The size range of the amplified DNAs was

between 100bp to 8,000bp as obtained by comparing with 1kb Marker DNA. Genetic variation among

population was estimated using the POPGEN 32 software package, VERSION 1.31.RAPD bands

separated on agarose gels were scored and transformed into a binary matrix where presence of band was

scored as 1 while absence of band was scored as 0. The mean observed number of alleles was estimated to

be ±2.000 while effective number of alleles was 1.5942±0.3148.

Plate 4.2.1 Gel Photograph of isolated genomic DNA

20

Plate 4.2.2 RAPD profile generated by primer OPA02


21


Table 4.2.1 Unique PCR bands amplified using different RAPD Primers

Primer 5’ to 3’ GC Content Unique Band Size (bp)

OPA 02 TGCCGAGCTG 70% 700, 1000

OPA 10 GTGATCGCAG 60% 400, 1300, 1600

OPA 11 AGGTGACCGT 60% 500

Table 4.2.2 Binary Data Input for POPGEN

K1 K2 K3 K4 K5 K6 K7 K8 O1 O2 O3 O4 O5 O6 O7 O8 02/200 1 1 0 0 0 0 0 0 0 0 0 0 0 0 1 1 02/300 0 0 1 1 1 1 0 0 0 0 0 0 1 1 0 0 02/400 0 0 0 0 0 0 1 1 1 1 0 1 0 0 0 0 02/500 1 1 0 0 0 0 0 0 0 0 0 0 0 0 1 1 02/700 0 0 1 1 1 1 1 1 0 0 0 0 1 1 0 0 02/1000 0 0 0 0 0 0 0 0 1 0 0 1 0 0 0 0 02/1500 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 02/1600 0 0 0 0 0 0 0 0 0 1 1 1 0 0 0 0 02/1800 0 0 0 0 0 0 0 0 1 0 0 0 1 0 0 0

22

02/5000 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 10/100 1 1 1 0 0 0 0 0 1 1 1 0 1 0 0 1 10/300 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 10/400 0 0 0 0 0 0 0 0 1 1 1 0 1 0 1 1 10/800 0 0 0 0 0 0 0 0 0 0 0 1 0 1 0 0 10/1000 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 10/1300 0 0 0 0 0 0 0 0 1 1 1 0 1 1 0 0 10/1400 1 1 1 1 1 1 1 1 0 0 0 0 0 0 0 0 10/1600 1 1 1 1 1 1 1 1 0 0 0 0 0 0 0 0 18/300 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 0 18/500 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 18/700 0 0 0 0 0 1 1 1 0 0 0 0 0 0 0 0 18/1200 0 0 0 1 1 1 1 0 1 0 0 0 0 0 0 0 18/1400 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 18/1500 0 0 0 1 1 1 1 0 0 0 0 0 0 0 0 0 18/1600 1 1 1 0 0 0 0 0 1 1 1 1 1 1 0 1 18/1800 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 18/2000 1 1 1 1 1 0 0 0 1 1 1 0 0 1 0 1 18/3000 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 1 18/8000 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 1

RAPD Data for POPGEN

/*Diploid RAPD Data Set*/

Number of populations = 16

Number of loci = 29

Locus name :

OPA02-200 OPA02-300 OPA02-400 OPA02-500 OPA02-700 OPA02-1000 OPA02-1500 OPA02-1600

OPA02-1800 OPA02-5000

OPA10-100 OPA10-300 OPA10-400 OPA10-800 OPA10-1000 OPA10-1300 OPA10-1400 OPA10-1600

OPA18-300 OPA18-500 OPA18-700 OPA18-1200 OPA18-1400 OPA18-1500 OPQ18-1600 OPA18-

1800 OPA18-2000 OPA18-3000 OPA18-5000

name = K1

10010000001000001100001010110

name = K2

10010000001000001100001010110

name = K3

01001000001000001100000010100

23

name = K4

01001000000000001100010101100

name = K5

01001000000100001100010101100

name = K6

01001000000100001100110100000

name = K7

00101000000000001100110100000

name = K8

00101000000000001100100000000

name = O1

00100110111010010011010010101

name = O2

00100001001010010011000010100

name = O3

00000001001010010011000010100

name = O4

00100101000001100011000010000

name = O5

01001000101010010011000010000

name = O6

01001000000001010011000010100

name = O7

10010000000010000011000000000

name = O8

10010000001010000001000010111

24

Table 4.2.3 Allelic Frequency of Each Locus

Locus Allele 0 (qi) Allele 1 (pi)

02/200 0.7500 0.2500

02/300 0.6250 0.3750

02/400 0.6875 0.3125

02/500 0.7500 0.2500

02/700 0.5000 0.5000

02/1000 0.8750 0.1250

02/1500 0.9375 0.0625

02/1600 0.8125 0.1875

02/1800 0.8750 0.1250

02/5000 0.9375 0.0625

10/100 0.5000 0.5000

10/300 0.8750 0.1250

10/400 0.6250 0.3750

10/800 0.8750 0.1250

10/1000 0.9375 0.0625

10/1300 0.6875 0.3125

10/1400 0.5000 0.5000

10/1600 0.5000 0.5000

18/300 0.5625 0.4375

18/500 0.5000 0.5000

18/700 0.8125 0.1875

18/1200 0.6875 0.3125

18/1400 0.8750 0.1250

18/1500 0.7500 0.2500

18/1600 0.3750 0.6250

18/1800 0.8750 0.1250

18/2000 0.3750 0.6250

18/3000 0.8125 0.1875

18/8000 0.8750 0.1250

25

Allele frequencies are calculated based on the frequency of the null allele (i.e., the number of individuals

without the band, or visually absent band) and the dominant allele (or visually present band; this does not

imply that the presence of a band is dominant over the absence). The presence of a band indicates either a

heterozygote or a homozygote for the dominant allele.

qi = individuals for which the band was NOT present

total individuals surveyed

pi = 1- qi

where qi represents the frequency of the null allele, and pi represents the frequency of the dominant allele

(Wallace, 2003).The above Table 4.2.3 is the result revealed and summarized by POPGEN 32 software

package, VERSION 1.31.

Table 4.2.4 Summary of Genetic Variation Statistics for All Loci

Locus Sample Size na* ne* h* I*

02/200 16 2.0000 1.6000 0.3750 0.5623

02/300 16 2.0000 1.8824 0.4688 0.6616

02/400 16 2.0000 1.7534 0.4297 0.6211

02/500 16 2.0000 1.6000 0.3750 0.5623

02/700 16 2.0000 2.0000 0.5000 0.6931

02/1000 16 2.0000 1.2800 0.2188 0.3768

02/1500 16 2.0000 1.1327 0.1172 0.2338

02/1600 16 2.0000 1.4382 0.3047 0.4826

02/1800 16 2.0000 1.2800 0.2188 0.3768

02/5000 16 2.0000 1.1327 0.1172 0.2338

10/100 16 2.0000 2.0000 0.5000 0.6931

10/300 16 2.0000 1.2800 0.2188 0.3768

10/400 16 2.0000 1.8824 0.4688 0.6616

10/800 16 2.0000 1.2800 0.2188 0.3768

10/1000 16 2.0000 1.1327 0.1172 0.2338

10/1300 16 2.0000 1.7534 0.4297 0.6211

10/1400 16 2.0000 2.0000 0.5000 0.6931

10/1600 16 2.0000 2.0000 0.5000 0.6931

18/300 16 2.0000 1.9692 0.4922 0.6853

18/500 16 2.0000 2.0000 0.5000 0.6931

26

18/700 16 2.0000 1.4382 0.3047 0.4826

18/1200 16 2.0000 1.7534 0.4297 0.6211

18/1400 16 2.0000 1.2800 0.2188 0.3768

18/1500 16 2.0000 1.6000 0.3750 0.5623

18/1600 16 2.0000 1.8824 0.4688 0.6616

18/1800 16 2.0000 1.2800 0.2188 0.3768

18/2000 16 2.0000 1.8824 0.4688 0.6616

18/3000 16 2.0000 1.4382 0.3047 0.4826

18/8000 16 2.0000 1.2800 0.2188 0.3768

Mean 16 2.0000 1.5942 0.3475 0.5219

St. Deviation 0.0000 0.3148 0.1335 0.1566

* na = Observed number of alleles

* ne = Effective number of alleles [Kimura and Crow , 1964] = Ae=1/ pi2

* h = Nei's (1973) gene diversity = 1- pi2- qi

2

* I = Shannon's Information index [Lewontin, 1972)] = - pi ln(pi)

(where, pi is the frequency of each allele observed across all investigated loci and qi is the frequency of

each null allele across all investigated loci.)

Hence, using the POPGEN 32 software package, VERSION 1.31, Nei's (1973) gene diversity was

calculated to be 0.3475±0.1335 and Shannon’s Information index [Lewontin (1972)] for the populations

was estimated to be 0.5219±0.1566 which is summarized in Table. 4.2.4.

Also following important data was also obtained:-

The number of polymorphic loci =29

The percentage of polymorphic loci = 100.00

27

4.3 Discussions

Population genetic methods are employed to analyze genetic variation observed in the species. Molecular

markers are commonly used to characterize genetic diversity within or between populations or groups of

individuals because they typically detect high levels of polymorphism. Furthermore, RAPDs are efficient

in allowing multiple loci to be analyzed for each individual in a single gel run. This investigation is for

the study of genetic diversity and genetic relatedness in the absence of knowledge of ancestry of all

individuals in the populations. Measure of various coefficients can be applied to quantify genetic diversity

and genetic relatedness among sample populations.

Gene diversity refers to the diversity for alleles (ultimately due to differences in DNA sequence) for a

specific gene, or locus, on a chromosome. Quantitative measures of gene diversity are based on the

number of alleles per locus, effective number of alleles per locus, the frequencies of alleles at each locus,

percentage of polymorphic loci, Nei’s (1973) gene diversity (h) and Shannon’s index(I). Diversity

increases as the number of alleles increases, and as the frequency of alleles becomes more evenly

distributed.Shannon's index is frequently used for RAPD studies becausethe index is insensitive to bias

that may be introduced intodata due to undetectable heterozygosity.

4.3.1 Correlation between Similarity Coefficients

Several measures are employed in the analyses of diversity of individuals. Two or three similarity

coefficients are used with the same data set with the expectation that if the results are robust, the different

coefficients should reveal essentially the same patterns of diversity. Though, similarity coefficients are

defined differently and therefore they may yield different results the correlation analysis can be carried

out to detect possible relationships between the different methods used. Correlation coefficient (r) is a

measure of the strength of the association between the two variables. Positive correlation indicates that

both variables increase or decrease together, whereas negative correlation indicates that as one variable

increases, so the other decreases, and vice versa.

In this investigation Nei’s gene diversity and Shannon’s index was estimated and Pearson correlation

analysis was carried out to detect possible relationships between the two different methods used (EXCEL

2007). There is then the underlying assumption that the data is from a normal distribution sampled

randomly (Figure 4.3.1.1). In conclusion, the result indicated that the strength of association between the

variables was very high, r = 0.992 and that the correlation coefficient was very highly significantly

different from zero with P < 0.001.

28

Figure 4.3.1.1 Normal Distribution Curve for Diversity Index

Figure 4.3.1.2 Graph for Pearson’s Correlation Coefficient, r = 0.992

29

4.3.2 Population Genetic Analysis

Table 4.3.1.1 Nei's Unbiased Measures of Genetic Identity and Genetic distance

Nei's genetic identity (above diagonal) and genetic distance (below diagonal).

From Table 4.3.1.1 revealed by POPGEN 32 software package, VERSION 1.31, we can together

summarize the genetic identity and genetic distance between the samples studied.According to the table,

genetic distance between pop 1 and pop 2 (both from Krak) was found to be 0, which suggests that these

two populations are genetically identical and hence shared the highest genetic similarity of 1. Highest

genetic distance was seen between pop 6 and pop 9 (both from Ongul) i.e., 1.1701. These two also shared

least genetic similarity i.e.0.3103.They both were from the same forest area but did not show much

genetic similarity. These results indicated that there are some differences between grouping by RAPD

markers and morphological traits.

The genetic distance among the Taxus populations from Krak ranged from being 0 (pop 1 and pop 2) to

0.5947 (pop 1/pop 2 and pop 6) while that among the samples from Ongul ranged from being 0.0351 (pop

10 and pop 11) to 0.5947 (pop 9 and 15). Pop 1/ pop 2 from Krak and pop 16 from Ongul shared the

highest genetic similarity of 0.7931. Pop 16 also shows least genetic distance with pop 1/pop 2 when

compared with other Krak samples. One reason for the high genetic similarity among them could be the

geographic proximity (Francisco-Ortega et al., 1993; Hormaza et al., 1994). Pop 16 of all the Ongul

samples was at the nearest distance (50m.) with pop 1 and 2 of Krak. Loveless and Hamrick (1984)

explained that populations in close neighborhoods tend to be uniform because genetic differentiation is

30

often prevented by gene flow. However, Pop 9 from Ongul shows highest genetic distance and shares

least genetic similarity with the samples from Krak.

To make the analysis more prominent, similarity and cluster analysis were conducted using Nei’s

coefficient and the Unweighted Pair Group Method with Arithmetic mean (UPGMA).

Figure 4.3.2.1 Dendrogram Based Nei's (1978) Genetic distance: Method = UPGMA

In the Dendogram, we can see that the Taxus populations from Krak and Ongul are chained separately. In

case of samples from Krak, pop 1 and pop 2 are illustrated as single cluster chained by pop 3 as out

group. Similarly, pop 4 and pop 5 are illustrated as single cluster, chained by pop6 as out-group which are

altogether chained by pop 7 and pop 8 as another cluster. In case of the samples from Ongul, pop 10 and

pop 11 are illustrated as one single cluster chained by pop 13 and pop 14. All these individual clusters are

chained by pop 15 and pop 16 and finally by pop 9 and pop 12 as the out-group.

Pop1-8 taken from Krak is chained like a single bunch by chain 13, whereas pop 9-16 taken from Ongul

is chained into a single bunch by chain 14. This clearly indicates that these each bunch of 8 samples

belong to two separate locations; one is Krak that is at an altitude of 2455m and another one is the Ongul

that is situated at an altitude of 2550m. However, Chain 15 connects both bunches this indicates even

though they belong to two different locations they have some distant relations that may be executed due

to the minor altitude difference. The similarities and differences among the populations may be because

of movement of populations progressively by natural or artificial means like birds, wind and air leading to

transfer of the genetic property from one to another.

31

Chapter V

CONCLUSIONS

Understanding choices made by any species at a habitat level is intrinsic to detecting patterns in

ecological space. On the other hand, understanding how variations in micro site in turn affect the

populations of any species is important in terms of ecological time. This study hence compares population

structure of Taxus , a threatened medicinal tree endemic to Himalaya, across different habitat types and to

studies micro site preferences (over space) exhibited by the species. This work not only gives the real

picture of the Taxus but also reflect the condition of vegetation of the selected area.

In recent years, molecular markers such as RAPD paired populations markers have been confirmed as

efficient tools for estimating genetic variation among genotypes of any organism (Williams et al., 1990).

These markers have the potential to measure variation with good coverage of the entire genome. RAPD

markers have clearly resolved patterns of diversity of many diverse types of plant populations and

germplasm collections. Findings from the research confirm the power of RAPD markers to reveal genetic

relationships among species’ populations, specifically relating populations of unknown origin with those

having more complete documentation.

This investigation has given an account of the genetic diversity of Taxus cultivars. During the present

study, a total of 132 DNA fragments were amplified in using RAPD primers, giving an average of 8.25

alleles per genotype. Thus, more primers should be screened to give a conclusive remark on genetic

diversity. Even so, we have prescreened a set of OPA primers on just a few samples and rejected the low

performing ones, rather than using all the primers in all of the cultivars, which is wastage of time and

resources. Further experimental investigation can now be done using these set of useful primers on rest of

the samples collected from rest of the sites. This will give an account of genetic diversity of whole Nepali

Taxus cultivars, which can be used as complementary tool for breeding strategy, since diverse parental

combination are required for creating segregating progenies with maximum genetic variability for further

selection. For a laboratory like ours just beginning to use molecular markers, RADP is the cheapest and

easiest DNA method. Hence, RAPD markers can be efficiently used to characterize Taxus cultivars but

more molecular data is required for better understanding of genetic variability and subsequent

improvement of Taxus baccata varieties in Nepal.

32

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35

APPENDICES

Figure 1. Distribution of Taxus baccata in Nepal

Figure 2. Map showing Hariyo Ban Working Area and Manaslu Conservation Area

(Source: USAID funded Hariyo Ban Progam in Nepal – Website)

36

Figure 3.Sample Collection Site

Figure 4. Sample Twigs of Taxus ready for rooting

37

Figure 5. Treatment of Twig cuttings with Phytohormone

Figure 6. Result of Rooting by Mode I (Liquid Mode), No any signs of Root Growth

38

Figure 7. Result of Rooting by Mode II (Dry Mode), Distinct signs of Root Growth

Table 1.Optimal separation range of Agarose

Percentage Fragment Size Range (kb)

0.3 5-60a

0.6 1-20a

0.7 0.8-10

0.9 0.5-7

1.2 0.4-6

1.5 0.2-3

2.0 0.1-2

Pulsed-field gel electrophoresis is required for efficient resolution of DNA fragments>15kb.Source:

Sambrook, J., and Russell, D.W. 2001. Molecular Cloning: A laboratory Manual, 3rd edition, Cold spring

harbor Laboratory Press, Cold Spring harbor,NY.

39

Table 2. List of primers.

Primer 5’ to 3’ M.W. pmoles µg/tube

OPA-01 CAGGCCCTTC 2964 6015 18

OPA-02 TGCCGAGCTG 3044 5495 17

OPA-03 AGTCAGCCAC 2997 5194 16

OPA-04 AATCGGGCTG 3068 5090 16

OPA-05 AGGGGTCTTG 3099 5194 16

OPA-06 GGTCCCTGAC 3004 5743 17

OPA-07 GAAACGGGTG 3117 4627 14

OPA-08 GTGACGTAGG 3108 4894 15

OPA-09 GGGTAACGCC 3053 5160 16

OPA-10 GTGATCGCAG 3068 5090 16

OPA-11 CAATCGCCGT 2988 5533 17

OPA-12 TCGGCGATAG 3068 5090 16

OPA-13 CAGCACCCAC 2942 5495 16

OPA-14 TCTGTGCTGG 3050 5785 18

OPA-15 TTCCGAACCC 2948 5785 17

OPA-16 AGCCAGCGAA 3046 4712 14

OPA-17 GACCGCTTGT 3019 5656 17

OPA-18 AGGTGACCGT 3068 5090 16

OPA-19 CAAACGTCGG 3037 4990 15

OPA-20 GTTGCGATCC 3019 5656 17

Reagents for Genomic DNA Isolation:-

40

1. Lysing Buffer

Lysing Buffer was prepared by adding 1gm of 2% CTAB, 0.78gm of 100mM Tris-HCl, 4.025gm of

1.4M NaCl, 1gm of 2% PVP and 0.292gm of 20mM EDTA to 40 ml distilled water. The pH was

adjusted to 8.0 using pH meter. The final volume was made up to 50ml, autoclaved and stored at

room temperature.

2. Chloroform: Isoamyl Alcohol (24:1)

3. Isopropanol (-20 C)

4. 70% Ethanol (Chilled, 4 C)

5. 96% Ethanol (Chilled, 4 C)

6. 5M Sodium Acetate (20.51gm in 50ml)

5M Sodium Acetate was prepared by dissolving 20.51gm sodium acetate in 50ml distilled water.

Solution was autoclaved and stored at room temperature.

7. TE Buffer

0.07gm of 10mM Tris-HCl, 1.43gm of 0.5M NaCl and 0.014gm of 1mM EDTA was added to 40 ml

distilled water. The pH was adjusted to 8.0 using pH meter. The final volume was made up to 50ml,

autoclaved and stored at room temperature

Reagents for Gel Electrophoresis

1. Electrophoresis buffer (TBE = Tris base, Boric Acid, EDTA)

5X stock solution of TBE Buffer was prepared by adding 54gm of Tris base, 27.5gm of Boric acid

and 20ml of 0.5M EDTA to 500ml of distilled water. The pH was adjusted to 8.0 using pH meter.

The final volume was made up to 1000ml, autoclaved and stored at room temperature.

2. Gel loading buffer

0.25% bromophenol blue, 0.25% xylene cyanol FF and 40% sucrose was mixed in distilled water to

prepared gel loading buffer.

3. Agarose

4. Ethidium bromide (0.5mg/ml)

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