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Species Differentiation in Tilia : A Genetic Approach

Thesis

to obtain Master of Science Degree in Tropical and InternationalForestry at the Faculty of Forest Sciences and Forest Ecology,

Georg-August-University Goettingen

By

Rajendra K.C.

Supervisors:

Prof. Dr. Reiner Finkeldey

Dr. Ludger Leinemann

Goettingen, Germany

January 2009

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To, My parents

Meena K.C. and

Chhatra Bahadur K.C.

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Acknowledgements iii

Acknowledgements

I am extremely grateful toProf. Dr. Reiner Finkeldey who has scholarly guided thisresearch throughout. Besides his scientific guidance, his parental support and caretoward my family at the difficult situation will always remain into our heart and soul.

I am very thankful to my co-supervisorDr. Ludger Leinemann who was alwayssupporting me with his scientific guidance. I was nurtured with his valuablesuggestions, feedbacks and comments throughout the work. I would like to share allcredits to my supervisors for the worthy findings and outputs in this study. However, Iwill be solely responsible for any errors or omissions.

I express my sincere thanks to Prof. Dr. Martin Ziehe and Dr. Elizabeth M. Gillet fortheir support in data analysis. I am thankful to Dr. Oliver Gailing for his support andguidance in conducting this research. The technical supports provided by ChristineRadler, Olga Artes, Oleksandra Dolynska, August Capelle and Gerold Dinkel can never be forgotten.

I would like to express my profound gratitude to Prof. Dr. Edzo Veldkamp, Prof. Dr.Christoph Kleinn, Prof. Dr. Ralph Mitlhner, Prof. Dr. Dirk Hoelscher and Dr. UweMuus for their continuous support and kindness.

I am also very thankful to Dr. Stefan Fleck, Jasmine Weisse, Nicole Legner, MeikMeiner and other colleagues for their support to conduct field works.

I would like to extend my sincere acknowledgement to Dr. Nicolas-George Eliades, Dr.Aye Bekele Tayele, Nga Phi Nguyen, Marius Ekue, Oleksandra Kuchma and Amaryllis

Vidalis for their assistance in many aspects of the research. I am also very thankful toMarita Schwahn for her tremendous support in providing favourable workingenvironment.

I am deeply indebted to our entire classmate (TIF 2006-2008) for their help, cooperationand friendship throughout the study period and establishing effective network for ourfuture endeavours.

I am very thankful to Prof. Dr. I.C. Dutta and Dr. Ridish Kumar Pokhrel (IOF,Pokhara); and Ram Prasad Paudel, Gopal Kumar Shrestha, Dr. Udaya Raj Sharma and

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Acknowledgements iv

late Dr. Damodar Prasad Prajuli of the Ministry of Forests and Soil Conservation fortheir constant support and inspiration to continue my academic career.

Our friends from Goettingeli Nepalese Society(GNeS) helped us in each and every

step of life. I am highly indebted to Netra Bahadur Bhandari, Lok Nath Paudel,Baburam Rijal, Dr. Ajaya Jang Kunwar, Rosan Devkota, Jeetendra Mahat, Dev RajGautam, Bharat Budthapa, Ajaya Pandey and Archana Guali for their great support. Iwould like to provide my sincere gratitude to Uschi Demmer (ChristophorusKindergarten) for her parental support to us.

I would like to thank Birgit Skailes and Stefan Heinemann(DAAD) for their instantsupport. They were so kind to us, we never required asking twice for any kind of helpthat we needed. I am deeply grateful to German Academic Exchange Service(DAAD) for providing me DAAD scholarship to study M.Sc. in Tropical and InternationalForestry (TIF). I am very thankful to Antze Henkelmann for her support throughout thestudy period.

I am very grateful to Ministry of Forests and Soil Conservation and Department ofForests, Nepal who supported me by granting study leaves.

I would like to offer all my indebtedness, gratitude and humbleness to my parents, brothers and sisters. My parents-in-laws also deserve special thanks for their continuoussupport and encouragement.

I am very thankful to my wife Aasha Khattri in flourishing the career and maintainingmy life. She never let me feel sad even in the most difficult situation in our family. Evenwhen our daughter Aarju was struggling for life, she tried to maintain entireenvironment favourable for my study. My children Ravi Raman, Avi Raman and Aarju

have been providing millions of smiles in my life despite I could not provide themenough time, sufficient care and better opportunities.

Rajendra K.C.Gttingen, 26 January 2009

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Table of Contents v

Table of Contents

ACKNOWLEDGEMENTS.................................................................................................III

TABLE OFCONTENTS..................................................................................................... VABBREVIATIONS..........................................................................................................VII LIST OFTABLES ..........................................................................................................VIII LIST OFFIGURES ...........................................................................................................IX APPENDIXES ..................................................................................................................X

1. INTRODUCTION............................................................................................... 11.1 Introduction to the Species ............................................................................... 1

1.1.1 The Family Tiliaceae .................................................................................... 11.1.2 The GenusTilia ............................................................................................ 1

1.2 Distribution ....................................................................................................... 21.3 Silivicultural Characteristics ............................................................................ 31.4 Morphology ...................................................................................................... 31.5 Phenology ......................................................................................................... 61.6 Use and Economic Importance of the Species ................................................. 6

2. LITERATURE R EVIEW .................................................................................... 82.1 Genetic Aspect ofTilia ..................................................................................... 82.2 Reproduction Biology inTilia .......................................................................... 92.3 Prevalent Methods of Species Identification inTilia ..................................... 10

2.3.1 Morphological Identification ...................................................................... 10

2.3.2 Genetic Identification ................................................................................. 132.3.2.1 Biochemical Marker ........................................................................... 132.3.2.2 Molecular(DNA) Marker .................................................................... 14

3. OBJECTIVES ANDR ESEARCH HYPOTHESIS ................................................ 173.1 Objectives ....................................................................................................... 173.2 Hypothesis ...................................................................................................... 17

4. MATERIALS ANDMETHODS......................................................................... 184.1 Materials ......................................................................................................... 18

4.1.1 Collection and Preparation of Samples ...................................................... 184.1.1.1 Study Area .......................................................................................... 184.1.1.2 Collection of Samples ......................................................................... 19

4.2 Methods .......................................................................................................... 204.2.1 Study of Nuclear Information ..................................................................... 20

4.2.1.1 Preparation of Materials ..................................................................... 204.2.1.2 Preparation of Buffer Systems ............................................................ 214.2.1.3 Preparation of Gels ............................................................................. 214.2.1.4 Loading the Samples .......................................................................... 224.2.1.5 Slicing the Gel .................................................................................... 234.2.1.6 Staining of the Enzymes ..................................................................... 234.2.1.7 Scoring the Zymogramme .................................................................. 244.2.1.8 Use of Enzyme Systems ..................................................................... 24

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Table of Contents vi

4.2.2 Study of cpDNA ......................................................................................... 264.2.2.1 Extraction of DNA ............................................................................. 274.2.2.2 Polymerase Chain Reaction ................................................................ 274.2.2.3 Agarose Gel Electrophoresis .............................................................. 28

4.2.2.4 Gene Scanning and Genotyping ......................................................... 304.3 Data Analysis .................................................................................................. 304.3.1 Analysis of Isozyme Data ........................................................................... 30

4.3.1.1 Species Assignment ............................................................................ 304.3.1.2 Determination of Genetic Pattern ....................................................... 34

4.3.2 Analysis of cpDNA Data ............................................................................ 354.3.3 Genetic Software for Data Analysis ........................................................... 36

5. R ESULTS........................................................................................................ 375.1 Interpretation of Isozyme Pattern ................................................................... 37

5.1.1 Menadione reductase .................................................................................. 375.1.2 Leucine Aminopeptidase ............................................................................ 385.1.3 Phosphoglucose isomerase ......................................................................... 385.1.4 Phosphoglucomutase .................................................................................. 405.1.5 Shikimate Dehydrogenase .......................................................................... 415.1.6 Alcohol Dehydrogenase ............................................................................. 415.1.7 Glutamate Oxalacetate Transaminase ........................................................ 42

5.2 Identification of Species ................................................................................. 435.2.1 Summer Linden (T. platyphyllos ) ............................................................... 445.2.2 Hybrid Linden (T. europaea ) .................................................................. 455.2.3 Winter Linden (T. cordata ) ........................................................................ 46

5.3 Genetic Patterns inTilia spp. .......................................................................... 475.3.1 Species-wise Genetic Pattern inTilia ......................................................... 495.3.1.1 Genetic Pattern ofT. platyphyllos ...................................................... 495.3.1.2 Genetic Pattern ofT. europaea ........................................................ 495.3.1.3 Genetic Patterns ofT. cordata ............................................................ 50

5.3.2 Genetic Pattern of Tilia spp. across Study Plots ......................................... 505.3.2.1 III-1 Plot ............................................................................................. 505.3.2.2 V1 Plot ................................................................................................ 515.3.2.3 V2 Plot ................................................................................................ 52

5.4 cpDNA Genetic Patterns ................................................................................ 535.4.1 Allelic Frequency ....................................................................................... 535.4.2 Haplotypes Frequency ................................................................................ 54

6. DISCUSSION ANDCONCLUSIONS.................................................................. 56

7. SUMMARY ..................................................................................................... 58

8. R EFERENCES................................................................................................. 61

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Abbreviations vii

Abbreviations

AAT Asparatate Amino Transferase

ADH Alcohol DehydrogenaseAP Aminopeptidase bp Base PairCCMP Consensus Chloroplast Microsatellite Primercm, mm, m Centimeter, Millimeter, MetercpDNA Chloroplast Deoxyribo Nucleic AcidDNA Deoxyribo Nucleic AciddNTP Deoxynucleotide Tri Phosphate

EDTA Ethylene Diamine Tetra Acetic Acid ( Na 2 Salt )GOT Glutamate Oxaloacetate TransaminaseGSED Genetic Structure from Electrophoresis DataHe Expected HeterozygosityHL Hybrid Linden (T. europaea )Ho Observed Heterozygosityml, l Millilitre, LitreMNR Menadione Reductase NAD Nicotinamide Adenine Dinucleotide NADH Nicotinamide Adenine Dinucleotide Hydrate ( Reduced Disodium salt ) NADP Nicotinamide Adenine Dinucleotide PhosphatePCR Polymerase Chain ReactionPGI Phospho Glucose IsomerasePGM PhospoglucomutasePPL Proportion of Polymorphic LociRF Relative FrequencySGE Starch Gel ElectrophoresisSKDH Shikimate DehydrogenaseSL Summer Linden (T. platyphyllos )SSR Simple Sequence RepeatsTAE Tris Acetate Ethylenediaminetetraacetic AcidWL Winter Linden (T. cordata )

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List of Tables viii

List of Tables

Table 1: Morphological traits to distinguish species inTilia ................................... 11

Table 2: Differences in morphological traits inTilia spp. ........................................ 12

Table 3: Enzymes used for isozyme electrophoresis ............................................... 26

Table 4: Expected size and position of cpDNA microsatellite primers .................... 26

Table 5: List of species specific alleles inTilia spp. ................................................ 31

Table 6: Identification of hybrid genotypes based on Mnr-A and Lap-D loci ......... 31

Table 7: The nomenclature of haplotypes produced by ccmp10 and ccmp3............ 35

Table 8: Grouping of cpDNA haplotypes according to species ............................... 44

Table 9: List of identifiedT. platyphyllos trees ........................................................ 44

Table 10: List of identifiedT. europaea trees ......................................................... 45

Table 11: List of identifiedT. cordata trees ............................................................... 46

Table 12: Distribution of haplotypes at study sites ................................................... 54

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List of Figures ix

List of Figures

Figure 1: Photograph of a Linden tree ......................................................................... 2

Figure 2: Photograph of various parts of Linden tree .................................................. 7

Figure 3: Map of Hainich National Park .................................................................... 18

Figure 4: Distribution ofTilia spp. inside research plots .......................................... 19

Figure 5: Photographs showing steps of isozyme electrophoresis ............................. 25

Figure 6: UV light photograph showing DNA amplification .................................... 27

Figure 7: Variations detected after PCR followed by agarose gel electrophoresis with

tested all universal primers ......................................................................... 28

Figure 8: Photograph after agarose gel electrophoresis of PCR products ................. 29

Figure 9: Electropherograms showing size variation in chloroplast microsatellitesccmp3 and ccmp10 ..................................................................................... 35

Figure 10: Photograph and schematic diagram of Mnr-A locus .................................. 37

Figure 11: Photograph and schematic diagram of Lap loci ......................................... 38

Figure 12: Photograph and schematic diagram of Pgi-loci .......................................... 39

Figure 13: Photograph and schematic diagram of Pgm loci ........................................ 40

Figure 14: Photograph and schematic diagram of Skdh loci ....................................... 41

Figure 15: Photograph and schematic diagram of Adh-A locus .................................. 42Figure 16: Photograph and schematic diagram of Got loci.......................................... 42

Figure 17: Frequency of identifiedT. platyphyllos ..................................................... 43

Figure 18: Allelic patterns across populations recorded from isozyme study ............. 47

Figure 19: Allelic frequency ofTilia spp. in all isozyme gene loci ............................. 48

Figure 20: Allelic frequency ofTilia spp. at III-1 plot ................................................ 50

Figure 21: Allelic frequency ofTilia spp. at V1 plot ................................................... 51

Figure 22: Allelic frequency ofTilia spp. at V2 plot ................................................... 52Figure 23: Relative frequency for alleles amplified with ccmp3 and ccmp10 ............ 53

Figure 24: Relative frequency of haplotypes inTilia .................................................. 55

Figure 25: Allelic patterns across populations recorded with cpDNA study ............... 55

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Appendixes x

Appendixes

Appendix 1: Recipe for preparation of homogenization buffer .................................. 69

Appendix 2: Recipe for preparation of gel buffer ....................................................... 69

Appendix 3: Recipe for preparation of electrode buffer ............................................. 70

Appendix 4: Recipe for preparation of starch gel ...................................................... 70

Appendix 5: Recipe for preparation of staining buffer .............................................. 71

Appendix 6: Electricity applied for electrophoresis .................................................... 73

Appendix 7: The protocol for running PCR for cpDNA ............................................ 73

Appendix 8: Isozyme and cpDNA data for individual trees at III-1 .......................... 74

Appendix 9: Isozyme and cpDNA data for individual trees at V1.............................. 75

Appendix 10: Isozyme and cpDNA data for individual trees at V2 ............................. 78

Appendix 11: Allelic structure ofT. cordata ............................................................... 81

Appendix 12: Allelic structure ofT . europaea .......................................................... 82

Appendix 13: Allelic structure ofT. platyphyllos ....................................................... 83

Appendix 14: Genotypic structure ofT. cordata ......................................................... 84

Appendix 15: Genotypic structure ofT. europaea ...................................................... 85

Appendix 16: Genotypic structure ofT. platyphyllos .................................................. 86

Appendix 17: Genetic parameters forT. platyphyllos .................................................. 87Appendix 18: Pairwise population matrix of genetic distance forT. platyphyllos ...... 87

Appendix 19: Genetic parameters forT. europaea ..................................................... 87

Appendix 20: Pairwise population matrix of genetic distance forT. europae a .......... 88

Appendix 21: Genetic parameters forT. cordata .......................................................... 88

Appendix 22: Pairwise population matrix of genetic distance forT. cordata .............. 88

Appendix 23: Various genetic parameters forTilia spp. at III-1 .................................. 89

Appendix 24: Pairwise population matrix of genetic distance at III-1 ......................... 89Appendix 25: Various genetic parameters forTilia spp. at V1 ..................................... 89

Appendix 26: Pairwise population matrix of genetic distance at V1 ............................ 90

Appendix 27: Various genetic parameters forTilia spp. at V2 ..................................... 90

Appendix 28: Pairwise population matrix of genetic distance at V2 ............................ 90

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Introduction 1

1. Introduction

1.1 Introduction to the Species

1.1.1 The Family Tiliaceae

Tiliaceae is one out of nine families under the order Malvale. Watson and Dallwitz

(1992) have extensively described the characteristics of the family Tiliaceae whichincludes trees, shrubs and in some cases herbs too.

This family has various leaves forms; mainly alternate, spiral or disctichous, petiolate,

non sheathing, stipulate and simple leaves. Plants under this family constitute mostly

hermaphrodites, or monoecious, or polygamomonoecious (Pigott, 1991; Watson and

Dallwitz, 1992). It might have either solitary flowers or aggregated in inflorescence

(Pigott, 1991). Flower contains a large number of androecium (15-100), free of one

another or coherent, and has stylate gynoecium (Pigott, 1991; Watson and Dallwitz,

1992). It produces fleshy or non fleshy; dehiscent or indehiscent or schizocarp fruits

(Watson and Dallwitz, 1992). It has endospermic seeds and its endosperm is rich in oil

content.

The family has around 450 species from 50 different genera (Watson and Dallwitz ,

1992). The plants under this family have world wide distribution. These species are

primarily distributed from tropical, subtropical to the temperate regions (Pigott, 1991).

1.1.2 The GenusTilia

Many of the species are found hybridizing in wild and in cultivation. Therefore, it is

difficult to identify the exact number of species under this genus, however it iscommonly agreed that the genusTilia has around 35-50 species (rodon, 1991). Thegenus includes the species, deciduous and tall trees, mostly native to northern

hemisphere. The species under this genus have been distributed to Europe, America and

Asia.

In this study, we will focus on Small Leaved Linden (Tilia cordata ), Large Leaved

Linden (Tilia platyphyllos ) and their putative hybrid (Tilia europaea ).

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Introduction 2

The taxonomical classification of theTilia spp. is as

follows:

Kingdom : Plantae

Subkingdom : TracheobiontaDivision : Magnoliophyta

Class : Magnoliopsida

Order : MalvalesFamily : Tiliaceae

Genus : Tilia Species: T. cordata Mill.

T. platyphyllos Scop.T. europaea L. and many other species.

Local name: Linden tree (Deutsch), Lime tree (English)

T. cordata and T. platyphyllos and their hybrid (T. europaea ) are the large trees of

various forms. It can grow up to 40 meter high, with a fairly cylindrical trunk up to 1

meter diameter at breast height. It produces clear bole up to two thirds of its height. The

trees on woodlands grow taller than open areas however the branching in the trees at

open areas start nearer to ground than woodland.

1.2 Distribution

Tilia species are widely distributed in Europe. Pigott (1991) has extensively studied the

geographical distribution ofTilia species. Among many species ofTilia , T. cordata is

one of the most widely distributed trees of Summer green deciduous forests of the

temperate low land of Europe and a small part of Western Siberia (Pigott, 1991). Itsdistribution is sub oceanic to sub continental.T. cordata reaches maximum elevation in

the Alps at 1450m whereasT. platyphyllos has a range in central and southern Europe,

and the most elevated stands have been reported also from the Alps at 1800m

(Boratynska and Dolatowski, 1991). The distribution range ofT. platyphyllos is quite

limited in comparison toT. cordata (Jensenet al., 2008) however both ofTilia spp. are

distributed all over Europe, in different proportion (Green, 1955; Pigott, 1991;rodon,1991; Chytryet al. , 1997; Wicksell et al. , 1999; cler and Mollamehmetoglu, 2001;

Figure 1.A Linden Tree

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Introduction 3

Jensen et al ., 2008). Due to its broader distribution in Europe, it can be referred as true

European broad leaved tree species.

Linden tree has long traditional, cultural and historical importance in many countries in

Europe as it has widely distributed in the region. For example it is regarded as thenational tree of Czech Republic (www.chez.cz) and Slovakia.

1.3 Silivicultural Characteristics

Linden trees generally occur as an associate of other species therefore rarely constitute

pure continuous forest. Lindens are deciduous trees which shed leaves during winter as

an adaptation strategy to severe cold.T. cordata is exceptionally tolerant to very low

temperature. It has been recorded undamaged after exposure to an air temperature of -

48C (Pigott, 1991).

Seedlings and saplings are relatively shade tolerant therefore they can survive even in

the frosty regions.T. cordata is lesser tolerant to shade thanT. platyphyllos (Pigott,

1991). T. platyphyllos is found at the intermediate stage whereasT. cordata is more

advance and found at the intermediate-climax stage of succession (Eriksson, 2001).

T. cordata, T. platyphyllos and their hybrids have remarkable capacity for vegetative

reproduction. They are good coppicers. Trees of all ages used to produce vigorous

shoots (coppices). Deer, sheep and cattle generally browse on their seedlings and

saplings (Pigott, 1991); therefore it is difficult to establish Linden trees in forest areas

with high game density.

1.4 Morphology

1.4.1 Crown

It has normally emergent and semi hemispherical crown spreading up to 5-12 m in

diameter. Old trees may form parabolic crown and reach up to 10-15 m in diameter.
http://www.czech.cz/sight-seeing/north-bohemia/semtin-linden-national-tree/http://www.czech.cz/sight-seeing/north-bohemia/semtin-linden-national-tree/http://www.czech.cz/sight-seeing/north-bohemia/semtin-linden-national-tree/http://www.czech.cz/sight-seeing/north-bohemia/semtin-linden-national-tree/

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Introduction 4

1.4.2 Branch

The lower branches in Linden mostly have horizontal and arching shape whereas

middle or second order branches usually have horizontal, ascending or vertical forms.

The upper layer branches are generally ascending and vertical.

1.4.3 Bark

Its bark is smooth, greyish with rhombus lenticels at the young age. The lenticels grow

into shallow fissures with increasing age and finally lie in deep fissures. The bark is still

thin but very firm and has scales on the ridges.

1.4.4 Leaves

Linden trees have simple, distichously arranged and palmately veined leaves. Leavesare alternate in two opposite rows. It has a long and slender petiole with a stipule. The

petiole ofT. cordata is slender and ranges from 0.8 to 1.0 mm in diameter and 3-6 cm in

length which is generally 0.5 to 2 times longer than lamina (Pigott, 1991).

The leaf is abruptly acuminate at the tip but its base is cordate or rarely truncate. It has

dentate margin on entire lamina except on the base. Leaves at the illuminated part of the

crown are flat and thicker than shadowed leaves. Leaves at the basal sprouts are

exceptionally larger and differ from those of crown in shape, marginal teeth and

distribution of hairs (Pigott, 1991).

Leaves ofT. cordata are smaller than that ofT. platyphyllos in length and width of the

leaf blade and apex. HenceT. cordata is also called as Small Leaved Linden andT.

platyphyllo s as Large Leaved Linden.

1.4.5 Flower

All Linden flowers are hermaphrodite (Hildebrand, 1869; Pigott, 1991). They have

complete flowers with five valvate sepals, five pale yellowish petals, large numbers of

stamens with yellowish anthers and pistil with spherical ovary. The floral formula for

Tilia is K5 C5AG (5) (Fromm, 2001). The flower is dish shaped and 1.0 to 1.5 cmlarge in diameter. The inflorescence ofT. cordata is weakly erect while T. platyphyllos

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Introduction 6

1.5 Phenology

Linden sheds leaves during winter. Leaf defoliation starts from October. As a result,

trees become completely leafless during whole winter. Leaf buds start to swell in April

and trees are again full with expanded leaves from middle of the May.

Linden starts flowering normally at the age of 25-30 years (Bsgen and Mnch, 1929;

Pigott, 1991) in woodland, and several years younger in open areas. Linden trees bloom

generally in between June and July.T. cordata flowers 10-15 days later thanT.

platyphyllos and T. europaea (Pigott, 1991; Chalupa 2003). Fruits grow throughout

the month of August and mature at the end of September.

1.6 Use and Economic Importance of the Species

Tilia is very important for its aesthetic and cultural value as a part of urban

forestry and landscape management.Tilia and their hybrids are among the most

favourite avenue trees in Europe.

The inner bark or bast consists of long and tough fibres that once were used inthe production of cordage, mats, and clothing (Rowe and Blazich, 2008).

Tilia produces good lumber popularly known as white lumber or brass wood.

But it can not be ideally used as the construction materials since it is soft and

rots easily. As wood is soft, straight grained, even textured and easy to work, it

is famous for wood carving.

Due to good acoustic properties, the wood is widely used in manufacturing the

musical instruments such as electrical guitar, drum shells, piano keys and others.

It does not produce splinters hence it is considered as an ideal wood formanufacturing handle for various tools and items (Rowe and Blazich, 2008).

Tilia flowers have pleasant fragrance and produce large quantities of nectar.

Therefore , it is highly favoured by bee keepers for the production of honey.T.

cordata is famous for many medicinal uses. The leaves and flowers can be used

as the medicine against flu and cough.

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Introduction 7

Figure 2: Parts of Linden tree:a) A matured Linden tree b) Alternate leaves foliagec) A simple leafd) Rough bark on mature treee) Smooth bark on young treef) An inflorescenceg) A bract & h) A fruit

a

d

b

c

e

g

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Literature Review 8

2. Literature Review

2.1 Genetic Aspect ofTilia

A systematic description of genus Tilia is difficult in view of the polymorphism of

species and the presence of numerous hybrids. The most commonly given number of

species are 35-50 under this genus (Giertych, 1991).

All three studiedTilia spp. are polyploids. The occurrence of more than two sets of

homologous chromosomes in the nucleus is called as polyploidy. Polyploid types are

named according to number of chromosome sets in the nucleus. Stebbins (1950)

estimated that about one third of the angiosperms species are polyploids. Further, Grant(1981) estimated about half of all angiosperms are polyploids. The occurrence of

polyploidy in animals is very rare events. The occurrence of polyploidy is a mechanism

of evolution and speciation in organism (Wright, 1976; Schultz, 1980).

Chromosomes ofTilia are very small, almost ovoid (Giertych, 1991), about 1m long

and 0.5m wide (Dermen, 1932; Pigott, 2002).Tilia has the very unusual high basic

chromosome number of 41 (Wright, 1976). Most of theTilia species are diploid

(2n=84) and few of them are polyploids such as tetraploid, hexaploid and octoploid.Pigott (2002) found out eight species as diploid (2n=84), five as tetraploid (2n=4=164)

and one as octoploid (2n=8 =328) out of 14 analyzedTilia species.

T. cordata and T. platyphyllos are the hexaploids that might have been evolved due to

inter species hybridization in the remote pasts. The genusTilia has n=41 chromosomes,

whereas all its relatives have n=7 chromosomes (Wright, 1976). A hexaploid (6n=42)

Tilia was presumably produced from an n=7 ancestor and lost a chromosome to become

n=41 (Wright, 1976). Chimera may occur in nature and it used to be induced by low

temperature (Giertych, 1991).

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Literature Review 9

2.2 Reproduction Biology inTilia

Tilia species are hermaphrodite plants. The availability of all reproductive organs in a

flower increases the chances for selfing. Selfing occurs in both T. cordata and T.

platyphyllos (Giertych, 1991). Fromm (2001) estimated that 30.1% average selfing rate

in T. cordata .

Fromm (2001) made extensive study about the production of pollen inT. cordata. As

per his study, a Linden flower produces around 43,500 (3,430) pollens. If the mean

number of flower is considered as five then there will have around 200,100 pollens in

an inflorescence. A ten year old branch usually has around 445 inflorescences so single

Linden branch may produce up to 89 million pollens. In total the production of pollen is

lower than other temperate tree species such asCarpinus betulus (>95 mill.),Quercus

petrea (>110 mill.) and Acer pseudoplatanus (>336 mill.) (Pohl, 1936; Fromm, 2001).

The pollen size ofT. cordata is estimated as 30 m (Fromm, 2001). The sink rate of

pollen flow is 3.2 cm/second (Knoll, 1932; Fromm, 2001). Average pollen transport

distance is around 79 m forT. cordata (Fromm, 2001). The stigma ofTilia is receptive

up to 2-5 days in general but up to 7 days in the cooler weather (Fromm, 2001).

Linden largely used to be pollinated by insects. A large number of insects used to visit

the flower. Both types of insects, diurnal and nocturnal visit Linden flowers because the

flowers used to open throughout the day and night. The insects are the most important

pollinators inTilia species however the wind may also contribute in pollination.

During the flowering season of Linden, it is only tree species in its distribution range

that flowers. Linden flowers produce attractive fragrances due to the secretion of

chemicals such as Fernasols and others (Pigott, 1991). Fernasols act as the pheromonefor bee (Fromm, 2001). The large numbers of flowers, availability of lots of sugars such

as fructose, glucose and sucrose, and conspicuous pale-greenish bracts etc. are the main

reasons of attraction for large number of insects. Prominently bees, flies and moths are

the major pollinators for these species.

Seed sterility is a common phenomenon in Linden (Pigott, 1991) howeverT. cordata

yields much more viable seeds thanT. platyphyllos (Giertych, 1991). Seed germination

is poorer and irregular inTilia spp. due to their hard seed coat and immature embryo

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Literature Review 10

(cler and Mollamehmetoglu, 2001). Natural regeneration of Tilia from seed is very

difficult hence the species frequently fail to regenerate. Krknen (2000) explains about

poor reproduction of T. cordata in Finland. Fromm (2001) mentioned about the

incapability ofT. cordata to reproduce by apomixes.

The mating system varies from autogamous to xenogamous. (Giertych, 1991) has

mentioned about the existence of natural hybridization and introgression betweenT.

cordata and T. platyphyllos . If T. cordata (2n=6 =84) andT. platyphyllos (2n=6 =84)

are found sympatrically in a region, they used to take part in spontaneous hybridization

(Pigott, 1991; Fromm, 2001).

T. cordata fruits fall within a distance of 100 meters from parent trees (Pigott, 1991),

and the seed flow in T. platyphyllos is assumed to be equal due to the similar

morphological traits of seeds to that ofT. cordata . The limited distance flow of seeds

creates a condition that the second generation offspring ofTilia species used to grow

relatively closer to its mother plant. This situation provides better opportunities for the

formation of family structures and back crossing between offspring and parents.

2.3 Prevalent Methods of Species Identification inTilia

The species identification forTilia spp. is possible through the study of morphological

characteristics or genetic traits. In following chapters we will summarize theoretical

concepts and prevalent practices of species identification forTilia spp. based on

published literatures.

2.3.1 Morphological Identification

Pigott (1969); and Wicksell and Christensen (1999) studied extensively the hybrids andhybridization of T. cordata and T. platyphyllos in England and in Denmark respectively

on the basis of its morphological characteristics. Both studies have mostly relied on the

morphological characteristics of leaves to distinguish between both species and their

hybrids.

Wicksell and Christensen (1999) looked at leaf characteristics as the major source of

species identification forT. cordata, T. platyphyllos and their hybrid (T. europaea ).

Size of lamina, leaf venation, width of apex, petiole size, serration of teeth, presence of

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hairs on leaf and on petiole, its colour etc. were taken as the major basis of species

identification (see Table 1).

Table 1: Morphological traits to distinguish species inTilia

Traits T. cordata T. platyphyll os

Length of leaf including basal lobe 45-106 mm 53-144 mm

Length of leaf excluding basal lobe 40-91 mm 48-135 mm

Width of lamina 40-82 mm 40-113 mm

Width of apex 2-9 mm 3-15 mm

Length of petiole 25-62 mm 22-62 mm

Number of teeth per cm on the broadest part of leaf blade 3-7 3-5

Numbers of lateral veins of first order 4-6.5 6-10

Presence of hairs on upper and lower surface of leaf Glabrous Pubescent

Types of hairs Stellate (forked) Simple

Colour of hairs Reddish brown White

Colour of abaxial surface of leaf blade Glaucous Green

Lateral veins of 2nd and 3rd order on abaxial surface Not raised

Raised

13. Presence of hairs on petiole Glabrous Pubescent

14. Presence of hairs on twig Glabrous Pubescent

15. Inflorescence Obliquely erect Pendulous

(Adapted from Wicksell & Christensen, 1999)

Anderson (1949) and Pigott (1969) suggested morphological traits for the analysis of

natural hybridisation and species identification (see Table 2).

Andrew (1971) mentioned that the pollen ofT. cordata is characterized by smalleraverage size, finer reticulation, a rounder outline than that ofT. platyphyllos .

Chamber and Godwin (1971) studied the species identification based on the microscopyof Tilia pollen. They studied pollen structure to distinguishT. platyphyllos and T.

cordata and their putative hybridT. europaea. They found differences in size and

shapes of pollen grain, pollen walls and in size and structure of funnels.

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Table 2: Differences in morphological traits inTilia spp.

Traits T. cordata . T. europaea T. platyphyll os

Leaves

Largest leaves on secondorder shoots < 8 cm 8-10 cm >10 cm

Adaxial surface

Flat, tertiary veins notraised on abaxialsurface

IntermediateRugose, tertiary vein prominent on abaxialsurface

Hairs on adaxial surface No hairs Scattered hairs Many hairs

Abaxial surface Glaucous Intermediate GreenAxils of veins on abaxialsurface Brown hair Pale hairs No tufts of hair

Veins of abaxial surface Hairless Scattered hairs Very hairyHairs between veins ofabaxial surface No hairs Scattered hairs Hairy

Petiole

Diameter of petiole 1.5 mm

Hairs on petiole No hairs Few hairs Many hairs

Twigs

Hairs on young twigs No hairs Few hairs Many hairs

(Adapted from Pigott, 1969)

The morphological traits as described by Pigott (1969); and Wicksell and Christensen

(1999) provide clear indication about the prevalence of many morphological traits

which are common and overlapping in both species. The size ranges of all

characteristics mentioned in Table 1 and 2 are continuous and overlapping. Furthermorphological traits are highly influenced by environmental factors whereas genetic

markers are not influenced by the environmental interaction. These are the reasons why

we use genetic markers for species identification.

Therefore, identification of the species on the basis of genetic approaches has been

experimented. Isozyme and chloroplast SSR (cpDNA) markers were applied to identify

the species inTilia.

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2.3.2 Genetic Identification

Genetic identification ofTilia spp. can be done by using genetic markers. The genetic

trait or the phenotypic characters that can be unambiguously assigned to a set of

genotypes is called genetic marker. If the genetic trait is due to the one genotype, it iscalled gene marker (Gillet, 1999).

Genetic markers have contributed tremendously to the development of the discipline of

genetics to the advanced stage. Genetic markers are applied to study the genetics of all

kinds of organisms. It can be used in wild and domesticated population of any animal or

plant population. Genetic markers have been used to identify the genetic variation,

mating systems, evaluating the impact of management, identifying the species,

development of plant phylogeny and so on.

There are several genetic markers frequently used in Forest Genetics according to the

needs and objectives of study. For the wider use and application, the genetic marker

should possess basic characteristics such as: inexpensive to develop and use, unaffected

by environmental variation, polymorphic and highly reproducible.

There are several genetic markers that have been developed and used in different

chronological period. Among them biochemical markers and molecular markers has

been tremendously effective and in application since their origin to date.

2.3.2.1 Biochemical Marker

Monoterpenes and isozyme are major biochemical markers. Due to many reasons

isozyme markers gradually replaced monoterpenes (Whiteet al., 2007). Isozyme marker

will be described in detail as we applied it in the laboratory to distinguish species.

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Isozyme Marker

Isozymes are special kinds of proteins that act as catalysts in chemical reaction in the

organisms. They act to enhance the rates of chemical reactions without themselves

being consumed.

Isozymes are the enzymes with similar or even identical functions (Finkeldey and

Hattemer , 2007). Markert and Moller coined the term isozyme to explain the differentmolecular forms of an enzyme in a species that share a common catalytic activity

(Acquaah, 1992). The multiple molecular forms of isozymes are quite common in

organisms and these isozymes share a common catalytic activity irrespective of its

differential molecular forms. The isozymes are often tissue or cell specific. Each and

every isozyme has a particular role and function in the metabolic pathway.

Isozymes arise in nature due to genetic and epigenetic mechanisms. Mutations such as

chromosomal and genetic aberrations constitute genetic origin of isozymes whereas

physical and chemical alteration of polypeptides during translation is the source of

epigenetic isozymes in organisms.

Isozymes have been successfully applied in investigations of the genetics of a large

number of organisms since 1960s (Liengsiriet al., 1990; Weising et al. , 2005).The

characteristics such as inexpensive operational costs, comparatively simpler and easier

laboratory work, availability of standard protocols for larger number of plant species,

codominant expression, relatively higher level of polymorphisms etc. have made

isozymes popular and extensively used marker in population genetics (Liengsiriet al.,

1990; Acquaah, 1992; Gillet, 1999; Bergmann and Leinemann, 2000; Finkeldey and

Hattemer, 2007; Whiteet al., 2007). It has been generally used to explain pattern of

genetic variation within and among population, estimating mating systems and geneflow (Whiteet al., 2007).

2.3.2.2 Molecular (DNA) Marker

There are only a small number of different marker loci therefore genetic information

obtained from biochemical marker, such as Terpene or Allozyme, may not be sufficient

representation of genes throughout the genome (White et al., 2007). Thus, it may lead to

erroneous conclusion about their protection and management (Szmidt and Wang, 1991).

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The limitation of biochemical marker insisted scientists to work for the development of

molecular or DNA based genetic marker.

Geneticist had developed this marker, so they applied first in the beginning of 1980s

(Botsteinet al. , 1980; Whiteet al., 2007). Soon after the invention, molecular markerswere used in plant for genetic analysis (Szmidt and Wang, 1991). The invention of PCR

and other advanced technologies made the application of molecular marker easier and

popular. With the advancement of technology and knowledge, various molecular

markers and analyses have been developed and applied for many purposes. Whiteet al.

(2007) mentioned two general types of DNA markers:

a) Based on DNA-DNA hybridization such as Restriction Fragment Length

Polymorphism (RFLP).

b) Based on amplification of DNA sequences using the PCR such as Random

Amplified Polymorphic DNA (RAPD), Amplified Fragment Length

Polymorphism (AFLP), Microsatellite or Simple Sequence Repeats (SSR) etc.

Microsatellite markers used in the study will be described in following chapter.

Microsatellites:

Microsatellites are also called as Simple Repetitive Sequences (SRS), Simple Sequence

Repeats (SSR) or Short Tandem Repeats (STR) (Weisinget al., 2005). As the name

itself explains, SSR (or microsatellite) are sequences of tandem repeats. Hancock (1999)

defines microsatellite as sequence made up tandem repeats from one to six bases in

length which are arranged head to tail usually without interruption.

There are two widely used methods to categorise the microsatellite. The first one is

based on the number of nucleotide available in the motif and the second one is related tothe degree of perfectness of the array. As per the first method, the microsatellite is

prefixed with mononucleotide [motif with single nucleotide e.g. (A)n], dinucleotide

[motif with two nucleotides e.g. (CA)n], trinucleotide [motif with three nucleotides e.g.

(CGT)n] and quadri-nucleotide [motif with four nucleotides e.g. (CAGA) n] and so on.

On the basis of degree of perfectness of the array, it can be categorised into three types:

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i) Perfect/pure: single uninterrupted array of particular motif such as ...(AG) n

ii) Imperfect: not pure but interrupted by another bases within repeated motif suchas ...(TC) n A(TC) n

iii) Compound: intermingled with perfect and imperfect arrays of several motifssuch as ...(AT)n(GT) n

The microsatellites are most often found in non-coding regions (such as introns) and are

seldom found in coding regions (exons) of the genome (Hancock, 1999; Weising et al .,

2005). They occur in all eukaryotic genomes (Weisinget al ., 2005). Microsatellite

presents in both nuclear and organellor (chloroplast, mitochondria) genomes however it

occurs much lower frequencies in organellor than nuclear genomes (Weisinget al.,

2005; Whiteet al., 2007).

Microsatellite gene markers are highly polymorphic and codominant. The allele size can

be more correctly identified with the precision of 1 bp. It requires low quantities of

template DNA and is highly reproducible. These meritorious properties are the reasons

for the wide range of application of this gene marker (Pandey, 2005). But the

requirement of sophisticated equipments, technologies, chemicals and larger investment

limits its wider application.

Chloroplast Microsatellite:

Microsatellites are the regular constituents at chloroplast but their frequencies are

greater than mitochondria and much lower than nucleus. The chloroplast genome

undergo virtually no recombination therefore the cpDNA is uniparentally inherited

(Harris and Ingram, 1991; Rhret al., 1998; Weising et al., 2005; Finkeldey and

Hattemer, 2007). In the case of angiosperm, it is inherited mostly from mother plantwhereas in the case of gymnosperm from pollen parent (Weisinget al., 2005).

Therefore, it is transmitted only through seeds in angiosperm (Derero, 2007).

The cpDNA is highly conserved DNA (McCauley, 1995). Therefore, it displays lowintra specific variation (Weising and Gardner, 1999). These properties are very

important to separate unrelated species from the targeted one. Besides, chloroplast

microsatellite can be used in maternity or paternity inheritance, detection of

hybridization and introgression, analysis of phylogeny etc. (Weising et al. , 2005).

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Objectives and Research Hypothesis 17

3. Objectives and Research Hypothesis

3.1 Objectives

T. cordata and T. platyphyllos have been growing sympatrically in the region for long

period. There are the admixture ofT. cordata, T. platyphyllos and their putative hybrids

(T. europaea). We are trying to identify the species of these individual Linden trees

on the basis of genetic markers. The main objectives of this study are:

To distinguish individual trees toT. cordata , T. platyphyllos and their hybrid on

the basis of already identified species specific genetic markers.

To compare species group concerning their cpDNA variation and differentiation.

To assess the genetic patterns inTilia spp. (T. cordata , T. platyphyllos and T.

europaea ).

3.2 Hypothesis

The main hypotheses of the research are as follows:

1. There is no evidence that species admixture ofT. cordata , T. platyphyllos lead to

inter species hybrids in natural stands.

2. Effective gene flow occurs mostly in one direction in that the less frequent

species acts mainly as seed parents.

3. There is no evidence for species differentiation in cpDNA haplotypes.

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Materials and Methods 18

4. Materials and Methods

4.1 Materials

We analysed bud tissues of individual tree without prior knowledge about species

origin. Prior to genetic identification, we did not have any information regarding the

morphological traits of the selected trees.

4.1.1 Collection and Preparation of Samples

4.1.1.1 Study Area

The research sites are located at Hainich National Park in Western Thuringia, Germany.The park covers an area of 75 square km, dominated by mixed deciduous forest

(www.nationalpark-hainich.de). Beech ( Fagus sylvastica ) is the dominant tree species,

grown in association with Ash ( Fraxinus excelsior ), Linden (T. cordata , T. platyphyllo s

and T. europaea ), Hornbeam(Carpinus betulus ), Oak (Quercus robur ), Elm (Ulmus

glabra ), Maple ( Acer pseudoplatanus and A. platanoides ) etc. Species composition of

the forests in Hainich has not largely disturbed by forestry operations since 1960s as it

was the part of military reservation area (Meisner, 2006). The park has the 7.5 C and

700 mm mean annual temperature and rainfall respectively (Cesarzet al., 2007).

Figure 3: Map of Hainich National Park (Frechet al. , 2003, referred in Meisner, 2006)
http://www.nationalpark-hainich.de/http://www.nationalpark-hainich.de/http://www.nationalpark-hainich.de/http://www.nationalpark-hainich.de/

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The samples were collected from three research plots namely III-1, V1 (new) and V2

established by Albrecht von Haller Institute of Plant Sciences, University of Goettingen.

The area of research plots are 50m wide and 50m long. The mean coordinate of research

sites are X=4394963 and Y=5661873 for III-1; X=4394956 and Y=5661852 for V1

(new) and X=4396845; and Y=5662679 for V2 research plots. These sites are around

367 m above sea level. The horizontal distance between III-1 and V1 is estimated 1.84

km whereas the distance between V1 (new) and V2 is merely 0.21 km. Linden trees are

concentrated around the middle part in III-1 research plot, and this plot is largely

dominated by F. sylvastica . The plots V1 and V2 have homogenous distribution of

Linden trees. We collected samples at these plots.

Figure 4: Distribution of Tilia spp. inside research plots

(Source: https://ufgb989.uni-forst.gwdg.de/GK1086/)

4.1.1.2 Collection of SamplesSamples were collected in the second week of April, 2008. During this period,Tilia and

other associate species were completely defoliated. Twigs with 10-25 buds were

collected from the base of each tree for convenience. Collected twigs were labelled with

same number tagged on trees. In total samples from 332 trees were collected.

Legend

Other research sub plots

Fagus

Acer

Fraxinus

Carpinus

Tilia

III-1 V1

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Twig samples were kept into climate chambers at 7C. The bases of twigs were cut with

scissor to enhance vascular activities and the twigs with buds were dipped into water

bucket, which is kept inside refrigerator to use in longer period. For the DNA isolation

and subsequent tests, 4 to 5 buds were picked up, and put into 2ml Eppendorf tubes, and

these tubes were frozen into liquid Nitrogen (-176C) for 10 seconds to prepare frozen

materials which were stored into refrigerator at -20C.

4.2 Methods

Species assignment followed the methods suggested by Fromm (2001). He identified

the species specific markers and methods to distinguishT. cordata and T. platyphyllos

based on isozyme variation. WL and SL possess species specific alleles which permit adifferentiation of both species and identification of the hybrid (Fromm, 2001).

Following his methods, the identification of species was made. The Mnr-A and Lap-D

loci were mainly considered while differentiating the species. These gene loci show

species specific alleles/variants inTilia .

Identified groups of the pure species T. cordata and T. platyphyllos as well as hybrids

were analysed with cpDNA markers to investigate differentiation at maternally

inherited genetic information. The combination of both isozyme gene loci and cpDNA

information allow us to estimate the direction of gene flow in mixed stand of both

species. Both isozyme and cpDNA study methods are discussed in following chapter.

4.2.1 Study of Nuclear Information

We applied the manual developed by Fromm (2001) for the isozyme electrophoresis.

The starch gel electrophoresis was undertaken for identifying the species variants. Thesubsequent laboratory procedures for isozyme study are discussed in following chapters.

4.2.1.1 Preparation of Materials

The selection of plant materials and its phenological stage needs to be duly considered

for better results in isozyme study. Newly formed bud tissues and fresh leaves were

used as the plant material for isozyme electrophoresis. The newly grown leaves ofT.

platyphyllos from the botanical garden of the Goettingen University were used as the

reference material at every electrophoresis run.

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Around 1-2 cm newly burst leaves were minutely homogenized in the extraction buffer.

Green tissues were given priority to pale or brown. Soaking wicks with extracted

enzyme was better and more convenient from fresh and green materials. Getting better

extraction is the most important part of isozyme electrophoresis.

Different amounts, sizes of bud tissue or leaf tissue, with different amount of extraction

buffer, were tested in the beginning to optimize the material and protocol. Finally the

leaf tissues of 15-20 mm2 and ~10g weight were macerated in 2-3 drops

(approximately 100l) of extraction buffer. Special attention was paid to prohibit

contamination. The recipe of extraction buffer is given in the Appendix-1.

4.2.1.2 Preparation of Buffer Systems

The buffers impart electrical conductivity to the support media and enhance the

activities of protein molecules. The preparation of appropriate buffer systems is crucial

in promoting better electrophoresis to obtain better separation and resolution of bands.

The ionic strength of gel buffer should usually be lower than that of electrolyte

(Acquaah, 1992). The appropriate combination of gel and electrode buffers is the

important factor to affect the resolution of isozyme patterns. Electrode buffers were

regularly changed after two uses to maintain their ionic strength. The buffers could be

stored successfully for one to two weeks in the refrigerator (at ~ 7C).

The homogenization (extraction) buffer, electrode buffers and gel buffers were prepared

as per the recipe from Fromm (2001) with little modification. The compositions of

different buffer systems are given in Appendixes 1-3.

4.2.1.3 Preparation of Gels

We used hydrolysed potato starch from GERBU Company to prepare gel. Starch gel

imposes additional restrictions to the migrating molecules due to their molecular sieving

properties besides only due to pH. In addition to this advantage, the starch gel technique

is simpler and less expensive than other electrophoresis systems therefore it is the most

practiced media for isozyme electrophoresis.

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Three different kinds of running systems such as Ashton and Braden (also called Tris-

borate) pH 8.1, Histidine-citrate pH 6.2 and Tris-citrate pH 7.4 were used for starch gel

electrophoresis. The recipe for the preparation of gel is given in Appendix- 4.

4.2.1.4 Loading the Samples

The gel was cut lengthwise around 2 cm away from the edge to load the wicks. The cut

divided the gel into two strips of different size; the narrow strip and wide strip. The

narrow strip was placed towards cathodal side and wider strip into anodal side to

provide longer space to hold isozyme variants. The cut itself performed as the origin of

isozyme separation.

The cut space was widened around 1 cm to load the wicks. The filter paper wicks of 5mm width and 6 mm length were used to absorb the enzymes from crushed bud tissues.

Wicks were loaded vertically to the anodal strip of the gel. They were placed at the

regular interval of 1-1.5 mm. In between the group of ten wicks, around 3-4 mm wider

interval was maintained for the easy and undoubted identification of samples during

zymogramme scoring.

A wick soaked with the homogenate from knownT. platyphyllos was also loaded at the

end of all samples for comparison. A wick saturated with Bromophenol blue

(C19H10Br 4O5S) marker was loaded at the end to monitor the electrophoresis progress.

Altogether 32 wicks, 30 from samples, 1 each from control species and marker were

loaded to the gel of 13 cm24 cm0.8-1.0 cm size. After loading all wicks, the cathodal

strip was firmly pushed back against the anodal strip to eliminate any gap in between.

For the better results, there should be good contact between anodal and cathodal strips

of the gel.

Then the glass plate with loaded gel was placed on the cooler plate in the

electrophoresis box. The high temperature generated during electrophoresis might

degenerate enzymes, therefore it is required to cool down the plate. The temperature

was maintained at 7C - 8C during electrophoresis.

The gel was covered around 2 cm from each edge with electrode towel, one end dipping

in the electrode buffer and other end covering the gel. The towel piece saturated with

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electrode buffer was placed firmly to ensure good contact with gel surface. Then, the

gel with towel was pressed with glass plate.

The electrophoresis was completed in four hours. The electricity was provided as

mentioned in the Appendix 6.

4.2.1.5 Slicing the Gel

The plates with gel were taken out after completion of the electrophoresis. The cathodal

gel was taken away and disposed. All the edges of anodal strip without any

electrophoretic variants were cut and removed. The upper right corner of gel was

diagonally cut to identify the numbering of samples afterwards.

The gels were sliced with metal filament. At first, the gel was sliced around 1 mm thin

and discarded. The gel was later sliced into two from Tris-citrate and Ashton and

Braden systems and three from Histidine-Citrate system. The thickness of slices was

maintained around 3 mm for all gels.

4.2.1.6 Staining of the Enzymes

The gel slice is dipped into staining solution to view the position of enzyme. The

staining solution contains optimum quantity of the enzyme substrate, appropriatecofactors and a dye. The visible bands on the gel are the products of the enzymatic

reaction associated with the dye. The proper staining is crucial for rightly scoring the

zymogramme.

The charged enzymes used to migrate from the origin through the gel during

electrophoresis. Isozymes migrate to different positions on the gel depending on the

electrical charge, temperature and size of isozymes. Isozymes, in general, have a

different charge and/or size due to its different amino acids composition. The genetic

differences are revealed as mobility difference on the gel (White et al., 2007).

Gel slices were first dipped into pre-buffer for few minutes before pouring staining

solution. Flasks with well stirred staining solution were heated in microwave oven for

30 seconds. The warm staining solution was poured over slices in staining tray. Before

pouring the staining solution, pre-buffer was put back for later use.

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The time and temperature play vital roles for enzymatic reaction to the dye. If the gel

slice is left for longer than optimum time, there can be the formation of additional

zones. That is not due to the true loci. Some enzyme system reacts easily in the room

temperature whereas some needs incubation at microwave oven. The trays with slice

and staining solution were incubated into microwave oven at 37C temperature for 1-2

hours. The staining tray with ADH enzyme was covered with glass plate inside

incubator since it contains ethanol. Different staining solutions have different recipe to

prepare. The recipe for each staining solution is given in the Appendix-5.

4.2.1.7 Scoring the Zymogramme

The process of gathering information is termed as the scoring (Acquaah 1992). The

scoring of the bands in zymogramme is the final and critical step in isozyme

electrophoresis. The study and interpretation of the zymogramme needs to be made very

carefully, since many internal and external factors might influence correct reading of the

bands. The separation and resolution of the electrophoretic variant influences the study

of zymogramme.

The gel slices were scored immediately after completion of staining. The gel was not

drained before scoring. The fastest migrated zone is named as A zone and fastestmoved allele in alocus is named as 1 . Similarly the slower migrated enzymes or zonesor alleles were named in ascending alphabets and numbers. The systems and

observation made by Fromm (2001) was implemented in scoring the zymogramme.

4.2.1.8 Use of Enzyme Systems

Seven different enzymes under three different running systems were applied for the

study. The Ashton and Braden, Tris-Citrate and Histidine-Citrate running systems were

successfully applied. Only the GOT electrophorants could not be studied due to the lack

of sharp and clear bands. The description of enzymes and their running systems is

briefly mentioned in Table 3.

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f

a

d

h

b

e

c

g

Figure 5: Photographs showing the stepsof isozyme electrophoresis: a) Samples ofTilia spp. in a bucket b) Extraction of isozymec) Preparation of starch gelsd) Loading samples into the gele) Putting gel into electrophoresis boxf) Running isozyme electrophoresisg) Slicing the gel h) Staining gel slices

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Table 3: Enzymes used for isozyme electrophoresis

(Adapted from Fromm, 2001)

4.2.2 Study of cpDNA

Two universal chloroplast primers namely ccmp3 and ccmp10 were applied to study

maternally inherited chloroplast DNA. For this, the expected fragment size and

annealing temperature of the selected ccmp primers were taken from Weising and

Gardner (1999) for general idea about the sizes of amplified fragments after gene

scanning. The expected sizes of two tobacco cpDNA microsatellite are given in Table 4.

Table 4: Size and position of cpDNA microsatellites for ccmp3 and ccmp10

Primer Primer sequence alignment Tm Expected size

ccmp3 5 -CAG ACC AAA AGC TGA CAT AG-3 5 -GTT TCA TTC GGC TCC TTT AT-3

51.3c 112 bp

ccmp10 5 -TTT TTT TTT AGT GAA CGT GTC A-3 5 -TTC GTC GDC GTA GTA AAT AG-3

53.7c 103 bp

(Adapted fromWeising & Gardner, 1999)

Enzyme and Nomenclature GeneLocus

Structure Numberof Alleles

RunningSystems

Glutamate oxalacetatetransminase(GOT) / Aspartateamino transferase(AAT) E.C.2.6.1.1

Got-AorAat-A

Dimer 2 Ashton andBraden

Phosphoglucose-isomerase(PGI) E.C.5.3.1.9

Pgi-BPgi-C

Dimer 42

Ashton andBraden

Alcoholdehydrogenase(ADH) E.C.1.1.1.1

Adh-A Dimer 3 Tris-citrate

Phosphoglumutase (PGM) E.C.2.7.5.1

Pgm-APgm-D

Monomer 34

Tris-citrate

Leucine Aminopeptidase(LAP) E.C.3.4.11.1

Lap-BLap-D

Monomer 4 Histidine-citrate

Menadione reduktase(MNR) E.C.1.6.99.2

Mnr-A Tetramer 4 Histidine-citrate

Shikimatedehydrogenase(SKDH) E.C.1.1.1.25

Skdh-B Monomer 6 Histidine-citrate

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4.2.2.1 Extraction of DNA

The DNA was isolated from Tilia buds following the protocol Purification of total DNAfrom fresh plant tissue of DNeasy Plant minikit (Qiagen, 2006). These buds were stored

in a refrigerator at the temperature -20c before the extraction. All the necessary

chemicals and accessories to isolate DNA were provided by Qiagen.

Initially few samples composed of all threeTilia spp. were chosen to study the variation.

For these 15 samples from all three possible species and oneT. platyphyllos sample from

New Botanical Garden of the Goettingen University were isolated in the beginning. Later

the DNA was isolated from 123 samples out of 332.

Figure 6: UV light photograph showing DNA amplification isolated fromTilia spp. on1% agarose gel.

4.2.2.2 Polymerase Chain Reaction

Polymerase Chain Reaction (PCR) allows the short fragment of DNA to be selectively

multiplied for further analysis (Finkeldey and Hattemer, 2007) hence it makes possible to

obtain result even if small amount of DNA is available.

The DNA was initially amplified for the test with eight out of ten universal cpDNA

primers. Eight tested fluorescence labelled consensus chloroplast primers were ccmp2,

ccmp3, ccmp4, ccmp6, ccmp7, ccmp8, ccmp9, ccmp10. Six out of eight primers showed

the amplification products. Only the primers ccmp3 and ccmp10 showed fragment length

polymorphism. PCR amplification was carried out in Peltier Thermal Cycler (PTC-0200

version 4.0, MJ Research). To run the PCR, the methodology mentioned by Demesureet

al. (1995) was followed with small modification in annealing temperature and

denaturation time.

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The 15 l PCR mixture containing 7.5 l of Hot Star Master Mix from Qiagen, 2 l of

10ng DNA template, 2 l each of 5 pM forward and 5 pM reverse primers, 1.5 l of

HPCL H2O per sample were used for PCR.

Figure 7: The variation detected after PCR followed by agarose gel

electrophoresis with all tested universal primers inTilia spp.

The PCR was carried out with an initial activation (95C for 15 ); thirty five cycles ofdenaturation (94C for 1 ), annealing (50C for 1 ), and elongation (72C for 1 ); andfinal extension of (72C for 10 ) and infinite time at 16C before taking the PCR productout from thermocycler. Primer with Hex (Green) fluorescent dye was used in PCR

process.

After completion of the process, the PCR product was tested in 1.5 % agarose gel

electrophoresis. The purpose of this test is to select better primers that show successful

amplification and to identify the strength of PCR products for gene scanning.

4.2.2.3 Agarose Gel Electrophoresis

The 1% and 1.5% of agarose (w/v) was used for the electrophoresis to check the quantity

and quality of DNA and PCR products respectively. 4.5 grams of agarose was kept into

300 ml of 1TAE buffer in a Flask. The gel was cooked in microwave oven until the

agarose powder get complete dissolution. The dissolution of agarose powder can be

observed simply with unaided eyes. If there is any particle seen, it should be furtherheated for complete dissolution.

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Ethidium bromide was added as the staining solution into Flask. 9 l of ethidium

bromide was put into the Flask. Since the ethidium bromide is very poisonous, it was

handled always inside the defume hoods wearing the protective gloves and glass.

The gel was poured into the plexiglass plate according to the requirement of the size ofgel. The masking tape was fixed tightly around the plate to make outer boundary and

stop leakage. Plastic combs were fixed into the plate before pouring the gel solution. The

DNA or PCR products were loaded later into these slots for electrophoresis.

The gel was cooled in room temperature for half an hour. The properly cooled agarose

gel was put into the running buffer (1x TAE) in the electrophoresis chamber and plastic

combs were removed. The samples (DNA / PCR products) were pipetted carefully into

the slots. The standard control was pipetted at the end of slots. Immediate after

completion of loading, the electricity was provided. The current and voltage provided

has strong influence on the speed of electrophoresis and its resolution. Electricity with

100 Volt potential was constantly applied for half an hour. After completion of

electrophoresis the gel was photographed with UV lights.

Figure 8: Photograph after agarose gel electrophoresis of PCR products

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4.2.2.4 Gene Scanning and Genotyping

For analysis on capillary sequencer, the PCR products were diluted as per the strength of

electrophorised bands. On the basis of strength of PCR product, we decided the

appropriate dilution ration. The gene scanner has very sensitive capillaries. If veryconcentrated PCR product is used, it may damage the capillaries and if the PCR product

is too dilute, it will not give clear and interpretable result. For the gene scanning, we

multiplexed both primers (ccmp3 and ccmp10) at the same tubes to be analysed.

As per the strength of electrophorized bands, we prepared the mixture with 1:100 rations.

1 l of ccmp3 and 1 l of ccmp10 primer were mixed with 98 l of HPCL water. The

known internal size standard GS 500 Rox (fluorescent dye) from Applied Biosystems

was kept to the sample to compare the results afterward. Finally we loaded 14 l [2 l of

diluted PCR products and 12 l Standard] per sample to the ABI 3100 Genetic Analyser.

Before loading probes to Gene Scanner, the assay was loaded into the PCR machine for

short denaturation at 90C for 2 minutes. The assay was cooled down by dipping into the

ice for 5 minutes before loading it into the ABI Genetic Analyser. We used the ABI

Genetic Analyser 3100 with internal size standard fluorescent dye ROX (Gene Scan 500

ROX) from Applied Biosystems.

The individual alleles were analysed using Genescan version 3.7 (Applied Biosystems)

and genotyped using Genotyper 3.7 software.

4.3 Data Analysis

4.3.1 Analysis of Isozyme Data

4.3.1.1 Species Assignment

Species assignment followed the methods suggested by Fromm (2001). He identified the

species specific markers on the basis of genetic information. Following these methods,

the identification of species has been done.

Fromm (2001) mentioned about the presence of species specific alleles to recognize

particular species inTilia (see Table 5). Different species specific alleles / variants have

been distinguished while scoring the zymogramme. On the basis of presence or absenceof these particular variants/alleles, we distinguished the species and grouped them intoT.

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cordata or T. platyphyllos or their hybrids (T. europaea ). Species specific alleles or

variants are mentioned in Table 5.

Table 5: List of species specific alleles / variants inTilia

Locus Species specific alleles /variants

Remarks

SL WLPgi-B B4 B1, B2, B3 may occur in both speciesPgi-C C1, C2 may occur in both speciesPgm-A A1, A2 may occur in both speciesPgm-D D1 D2, D3 may occur in both speciesMnr-A A1, A2 A3, A4 Skdh-B B5, B6 B1, B2, B3, B4 may occur in both speciesLap-D D3, D4 D1, D2 Adh-A A1, A2, A3 A1, A2, A3 may occur in both species

The presence of alleles or variants from both species ofTilia in an individual tree was

undertaken as the hybrid. A tree is assigned F1 generation hybrid if all discriminating loci

show heterozygote genotypes combined with alleles from both species and a tree is

assigned advanced generation hybrid if one locus indicates as hybrid and other locus as

any of pure species (Table 6). The Mnr-A and Lap-D loci were highly considered while

assigning the species as they were discriminating and discrete among all analysed geneloci.

Table 6: The identification of hybrid generation based on Mnr-A and Lap-D loci

Species Mnr-A Lap-D Remarks

T. cordata AxAy DvDw Where,X (y)= 3 or 4V (w) = 1 or 2T. platyphyllos AvAw DxDy

1st

generation hybrid AvAx DvDx Advanced generation hybrid All other cases

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4.3.1.2 Determination of Genetic Pattern

4.3.1.2.1 Geneti c Structur e

Allelic Structure

Allelic structure can be explained as all frequency distributions of alleles of the

population at the respective marker gene locus. It can be calculated as either relative or

absolute frequency. The relative frequency of the alleles can be calculated as

pi =ni / 2N......in the case of diploid species.pi =ni / ni .in the case of polyploid species.

Where,

pi= Relative frequency of Allele Ai ni= Absolute frequency of Allele Ai

N= number of studied genotypen= number of alleles

Genotypic Structure

Genotype is the collective genetic information within an organism at the studied gene

locus. Finkeldey and Hattemer (2007) describe genotypic structure as the frequency

vector of all (relative) frequencies of genotypes.Pij=Nij / N

Where,

Pij= Relative frequency of genotype AiA j

Nij=Absolute frequency of genotype AiA j

N= Number of studied genotypes.

4.3.1.2.2 Proporti on of Polymorphi c Loci (PPL)

Proportion of Polymorphic Loci takes account only the occurrence of different genetic

types, but not their frequencies. It is calculated by dividing the number of polymorphic

gene loci by the number of all investigated loci including monomorphic loci.

PPL= PL /(PL+ML)Where,

PPL = Proportion of Polymorphic Loci

PL = Polymorphic Loci & ML = Monomorphic Loci

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4.3.1.2.3 Average Nu mber of Al leles per Locus (A / L)

It is calculated by dividing of total number of alleles counted by the number of loci

observed.

Average Number of Alleles= ni / L

Where,

ni= Number of alleles at locus iL= Total number of all observed loci

4.3.1.2.4 Genetic Di versi ty

Effective Number of Allele (Ne)The effective number of alleles explains about the distribution pattern of allele

frequencies. This is also related to the concept of expected heterozygosity. It reaches

maximum when the He is the maximum or when the distribution of all alleles are equal

in the population. Gregorius (1978) defined Ne as the allelic diversity (v) of the

population at the given gene locus (Finkeldey and Hattemer, 2007). This can be

calculated as

1 v = 1/ pi2 n i

Where, pi = Frequency of alleles in a locus

Observed Heterozygosity (Ho)

The observed heterozygosity can be explained as the proportion of all heterozygote

genotypes present in the studied population.

n n

Ho = Pij = 1-Pii i=1 j=1

Where,

Pij= Relative frequency of genotype AiA j (heterozygote)

Pii= Relative frequency of genotype AiAi (homozygote)

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Expected Heterozygosity (He)

Expected heterozygosity can be defined as the probability that may have heterozygotecombination from two alleles randomly drawn from individuals in a population.It is

most important and widely used parameters in comparing the genetic variation among population (Finkeldey and Hattemer, 2007). It is also called as the gene diversity.

The term He can be explained in terms of the allelic structure only, hence createsconfusion since the heterozygosity refers to the genotypic structure of the organism.

He can be obtained by deducting the sum of the frequencies of expected homozygosityfrom 1, as per the following formula:

He=1- pi2 iWhere,

He = Expected heterozygosity pi = Frequency of alleles in a locus

Fixation Index

Fixation index is a measure of population differential based on genetic polymorphism.

Fixation index provides the idea about genetic make up of actual population calculatedfrom estimated genotypic frequencies (Whiteet al., 2007).

The fixation index ranges from -1 to 1.The F=0 implies about the complete absence of

inbreeding and the perfect matches of genotypic frequencies with Hardy-Weinberg

principle. Whereas F=1 explains about the sole prevalence of inbreeding.

The formula to calculate fixation index is as follows

F= He-Ho / HeWhere,

Both He (Expected heterozygosity) and Ho (Observed heterozygosity) are themeans calculated from all analysed gene loci.

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4.3.2 Analysis of cpDNA Data

We have studied the cpDNA variation from 123 samples. The cpDNA provided genetic

information regarding the seed parents only. The ccmp3 and ccmp10 markers showedclear results at expected fragment size. At the Figure 9, the first graph shows the

fragments size 112bp by ccmp10 and 128bp by ccmp3. In the second graph, the ccmp10

and ccmp3 produced 113bp and 128bp fragment size of DNA respectively.

Figure 9: Electropherograms showing size variation in chloroplast microsatelliteccmp3 and ccmp10 as visualised by Genescan 3.7 and Genotyper 3.7

The linked fragment size loci obtained from the amplification with ccmp3 and ccmp10

were grouped into Haplotypes (Table 7).

Table 7: The nomenclature of haplotypes produced through the combination of

ccmp10 and ccmp3 primers

ccmp10ccmp3

103 bp 106 bp 112 bp 113 bp

127 bp 1 2 - -

128 bp - - 3 4

129 bp 5 - - -

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Results 37

5. Results

5.1 Interpretation of Isozyme Pattern

5.1.1 Menadione reductase

MNR is the tetrameric enzyme. Being a member of tetrameric enzyme system, it shows

low level of polymorphism than monomeric and dimeric enzyme. It produces only one

zone of activity. After proper staining, we can view clearly four alleles at this locus.

This enzyme system is very important for identification of species inTilia . It produces

the species specific alleles inT. cordata and T. platyphyllos .

Figure 10: Photograph and schematic diagram of Mnr-A inTilia

Alleles A3 and A4 are specific to Winter Linden (T. cordata ) whereas A1 and A2 are

specific to Summer Linden (T. platyphyllos). Mnr-A locus showing one allele from WLand another from SL, indicates that the particular tree is the Hybrid between these two

species. Genotypes A1A1, A1A2, A2A2 were specific to SL trees and A3A3, A3A4, A4A4were WL specific whereas A1A3, A1A4, A2A3 and A2A4 were recorded only in hybrid

Linden (T. europaea ).

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Results 38

5.1.2 Leucine Aminopeptidase

The sub cellular region of this monomeric enzyme is cytosol and this is used to be

activated by heavy metals. While staining, we used both leucine and alanine as per the

quantity mentioned in recipe at Appendix 5(F).

This enzyme system produced 4 different zones of activities however zone A and B

were observed blurred and zone C showed low variation. The D zone produces species

specific alleles, so that the Lap-D zone is very crucial while identifying the WL, SL and

their hybrids. D1 and D2 alleles are specific toT. cordata whereas D3 and D4 are specific

to T. platyphyllos . The heterozygous genotype containing either D1 or D2 with any of D3

or D4 alleles are considered as their putative hybrids (T. europaea ). Genotypes D1D1, D1D2, D2D2 were found in T. cordata whereas D3D3, D3D4, D4D4 were found in T.

platyphyllos trees. Genotypes D1D3, D1D4, D2D3 and D2D4 were observed only in

Hybrids.

Figure 11: Photograph and schematic diagram ofLAP loci inTilia

5.1.3 Phosphoglucose isomerase

The phosphoglucose isomerage is the dimeric enzyme, which is also known as

glucosephosphate isomerase or phosphohexose isomerase. It functions in glycolysis

(Freeling, 1983; Acquaah, 1992). The sub-cellular location of PGI is cytoplasm and

plastids (Gottlieb and Weeden, 1981; Acquaah, 1992). After electrophoresis PGIfrequently shows polymorphic variation in plants (Lacket al ., 1986).

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Results 39

PGI showed three different zones of activities. All the zones showed the variation

however due to blurred phenotypes and also unavailability of nomenclature keys, Zone

A could not be analysed.

5.1.3.1 Pgi-B

Overall four alleles were observed at this locus. The alleles B1, B2 and B3 were common

in T. cordata and T. platyphyllos . SL showed completely different zymogramme from

T. cordata at this locus. These different patterns are due to the presence of variant B4, but its confirmation of specific allele toT. platyphyllos must be tested by analysing the

larger number ofT. platyphyllos samples.

5.1.3.2 Pgi-CTwo alleles were observed at C-zone. This Pgi-C locus produced minimum variation

however the zymogramme pattern ofT. cordata and T. platyphyllos was different at this

locus too. This locus mostly used to be considered as the complementary to identify the

species in combination with other gene loci.

C1 allele for T. cordata and T. platyphyllos were phenotypically different hence

visualising it as different two alleles or variants. C1 allele of T. cordata was observed

faster and farther than that ofT. platyphyllos . So, many times SL variant is named as C2.

Figure 12: Photograph and schematic diagram ofPGI loci inTilia

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Results 40

5.1.4 Phosphoglucomutase

The sub cellular l

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