Is Alnus viridis 'a' glacial relict in the Black Forest? [Elektronische Ressource] / vorgelegt von Sultana Kamruzzahan

Sultana Kamruzzahan

Informations

• Publié par: albert-ludwigs-universitat_freiburg
• Publié le: 01 janvier 2004
• Nombre de lectures: 12
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

Is Alnus viridis ‘a’ Glacial Relict in
the Black Forest?









Inaugural-Dissertation Zur Erlangung der Doktorwürde der Fakultät für
Biologie der Albert-Ludwigs-Universität Freiburg im Breisgau





Vorgelegt von

Sultana Kamruzzahan
Master of Science in Botany, Bangladesh
December - 2003








Dekan: Prof. Dr. H. Kleinig


Leiter der Arbeit: Prof. Dr. P. Nick


Promotionsvorsitzender: Prof. Dr. K.F. Fischbach


Referent: Prof. Dr. P. Nick

Koreferent: Dr. A. Bogenrieder




Tag der Verkündigung des Prüfungsergebnisses: 25 February, 2004




















Dedicated to my husband and my son






















Acknowledgement

I am very grateful to Prof. Dr. Peter Nick for supervising this Ph. D. dissertation and for his interest and
support of this study which is greatly appreciated.

I am deeply indebted to Prof. Dr. Eckard Wellmann, Institute for Biology II, Botany, Universität
Freiburg, who allowed me to do a part of my thesis in his laboratory and made it possible to do an
extensive photomorphogenesis study of Alnus viridis by providing his laboratory facilities.

I am very greatful to Dr. Thomas Borsch, Botanisches Institut, Friedrich-Wilhelms-Universität Bonn,
who introduced me to molecular systematics in his laboratory and committed a lot of his time for
discussions about molecular questions.

I am also grateful to Prof. Dr. Arno Bogenrieder, Institute for Biology II, Geobotany, Universität
Freiburg, for providing help with the collection of material and valuable discussion. It is a particular
pleasure for me to thank Prof. Dr. Thomas Speck, Botanical Garden, Universität Freiburg, who has
been helping me with his long and outstanding experience with Alnus.

I would like to thank my husband Dr. Abdul Ahad and son Afif Ehasan Kabir to provide me always
mental support during this period. I greatly appreciate the consistent support by my parents, who shared
an interest in my work and were very motivating during all times.

I wish to express my warmst thanks to all members of Dr. Nick group for the always nice working
atmosphere and everybody's helpfulness.

My great thanks to Dr. Wolf Reutz, Bayerishes Amt für Saat -und Pflanzenzucht, Bavaria, and Dr.
Franz Schuhwerk, Botanische Staatssammlung, München, for effort to sending plant materials specially
for molecular work.

I am also greatful to the Fazit Stiftung, Frankfurt am Main for partial financial support to finish my Ph.
D. work








CONTENTS

1. INTRODUCTION…………………………………………………………..……….….….1

1.1 Ice ages and vegetation …………………………………………..……….....……1
1.2 Survival strategies of glacial relicts……………………………..……….……….2
1.3 A. viridis as a model for glacial relicts…………………………..…….…....…….3
1.4 Scope of the study…………………………………………………..……….……..7

2. MATERIAL AND METHODS…………………………………………………..….…….9

2.1 Material
2.1.1 Chemicals ………………………………………………….…..…..….…9
2.1.2 Kits for molecular biology……………………………..……..….……..10
2.1.3 Enzymes………………………………………………….…...…..……..10
2.1.4 Primers………………………………………………….…..…..……….10
2.1.5 Markers for DNA……………………………………….….…...………10
2.1.6 Accessories……………………………………………….……………...11
2.1.7 Light sources…………………………………………….…..….….……12
2.1.8 Plant material and sampling………………………………....…......….13
2.2 Methods
2.2.1 Photobiological studies……………………………………………..…16
2.2.1.1 Seed germination…………………………………..……..…...16
2.2.1.2 Irradiation condition……………………………..…….….…16
2.2.1.3 Hypocotyl growth………………………………….….……....16
2.2.1.4 Extraction of flavonoids from hypocotyls and cotyledons…16
2.2.1.5 Extraction of anthocyanin from hypocotyls and scale
leaves…………………………………………………………………..17
2.2.2 Molecular analysis
2.2.2.1 Isolation of DNA from plant leaves………………………….17
2.2.2.2 RNAse treatment……………………………………………...18
2.2.2.3 cpDNA regions and primers…………………………..….…..18
2.2.2.4 PCR amplification and DNA sequencing……………...…...19
2.2.2.5 Sequence alignment and indel coding……………………….20
2.2.2.6 Outgroup selection……………………………………………20
2.2.2.7 Molecular clock determination ……………………..…...…..21
2.2.2.8 Phylogenetic tree analysis……………………………….…....21

3. RESULTS
A. Photobiological studies………………………………………………….…….….23
3.1.1 Introduction……………………………………………….………….…23
3.1.1.1 Photoreceptors…………………………………….………..…23
3.1.1.1.1 Phytochromes…………………………………………….…24
3.1.1.1.2 Cryptochromes and phototropins………………………….25
3.1.1.1.3 UV-B receptor………………………………………...……..26
3.1.1.2 Synthesis of flavonoids/anthocyanin and hypocotyl
inhibition…………………………………………………………..…..26
3.1.1.3 Scope of the study……………………………………………...….…..28

3.1.2 Results………………………………………………………………….…30
3.1.2.1 Spectral dependence of hypocotyl inhibition…………..……..30
3.1.2.2 Spectral dependence of flavonoid formation…………….……30
3.1.2.3 Spectral dependence of anthocyanin formation……….……...36
3.1.2.4 Dark germination and scale-leaf anthocyanin differs in
the Black-Forest population……………………………………..36

3.3 Discussion……………………………………………………………...……38
3.3.1 What are the responsible photoreceptors in different
population? ………………………………………………………38
3.3.2 How do population differ due to the habitats differ?…….…....…41
3.3.3 What is the possible adaptive function of the observed
differences?………………………………………………………..42

B. Molecular Systematics of Green Alder……………………………….…………. 43
4.1 Introduction…………………………………………………………………..43
4.1.1 chloroplast genome is a genetic marker for genetic
variation………………………………………………………………….....…43
4.1.2 Chloroplast DNA (cpDNA) structure……………………….….....…44
4.1.3 Molecular clocks and the estimation of divergence times……..…...47
4.1.4 Scope of the study…………………………...………….……….……47
4.2 Results…………………………...…………..…………………………………48
4.2.1 Characterization of the trnT-trnF-region of the chloroplast
genome in Alnus……………………………………….…………..48
4.2.2 Analysis of the trnT-trnL intergenic spacer……………..………48
4.2.3 Analysis of the trnL intron………………..………..…….………49
4.2.4 trnL-trnF intergenic spacer….……..………….51
4.2.5 Grouping/clustering of the populations…………………….…55
4.2.6 Analysis of divergence times……………………………………56

4.3 Discussion…………………………………………………………………….. 59
Molecular evolution of trnT-trnF non-coding chloroplast region in Alnus..59
The trnL-trnF region is the most variable part of cpDNA in Alnus………..61
Are St. Blasien and Schulterdobel completely differ than others? ………..61
Why did the Alnus sequence not follow a general phylogenetic tree……....63

5. GENERAL DISCUSSION
Marker based study of phylogenetic relationship in Plant kingdom……...65
cpDNA sequence (trnT-trnF) and relationship with angiosperm……..…...66
Phylogenetic tree and genetic distance………………………..….……...…..67
Pollen fossil history and relationship with glacial relict of Alnus……….....68
A.viridis in the Black Forest not ‘a’, it is several glacial relict
populations…………………………….…………………........………………69

6. SUMMARY……………………………………………………………….….…...………..74

7. REFERENCES………………………………………………………………….………….76










List of abbreviations
R red light
FRfar-red light
B blue light
UV-A ultraviolet A (indicate the wavelength range)
UV-B ultraviolet B (indicat
EDTA Ethylendiamin-tetraacetic acid
PCR polymerase chain reaction
TRIS tris-(hydroxymethyl)-aminomethan
TE Tris-ethylendiamintetraacetic acid
RPM rotation per minute
kB kilobase
M molar
CTAB Cetyltrimethylammoniumbromide
bp base pair
Myr Million years









1
1. Introduction
1.1 Ice ages and vegetation
Ice ages are geological periods when large portions of land were covered by ice
(http://www.museum.state.il.us/exhibits/ice_ages). The earliest ice ages can be dated back to
Precambrian times (more than 600 million years ago), followed by a second ice age during the
early Cambrian (about 600 million years ago), and a thir