Genetic characterisation of populations from the European natural range of Norway spruce (Picea abies (L.) Karst.) by means of EST markers [Elektronische Ressource] / Maryna Valdivia Chevarria
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English

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Genetic characterisation of populations from the European natural range of Norway spruce (Picea abies (L.) Karst.) by means of EST markers [Elektronische Ressource] / Maryna Valdivia Chevarria

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Publié par
Publié le 01 janvier 2005
Nombre de lectures 12
Langue English
Poids de l'ouvrage 1 Mo

Extrait

Fachgebiet Forstgenetik




Genetic characterisation of populations from the European
natural range of Norway spruce (Picea abies (L.) Karst.)
by means of EST markers



Maryna Valdivia Chevarria


Vollständiger Abdruck der vom Wissenschaftszentrum Weihenstephan für Ernährung,
Landnutzung und Umwelt der Technischen Universität München zur Erlangung des
akademischen Grades eines
Doktors der Naturwissenschaften (Dr. rer. nat.)
genehmigten Dissertation.

Vorsitzender: Univ.-Prof. Dr. Gerhard Wenzel
Prüfer der Dissertation:
1. Univ.-Prof. Dr. Gerhard Müller-Starck
2. Univ.-Prof. Dr. Gert Forkmann

Die Dissertation wurde am 08.11.2004 bei der Technischen Universität München
eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung,
Landnutzung und Umwelt am 09.03.2005 angenommen.



Partial results of this dissertation were published as follows:

M. Bozhko, R. Riegel, R. Schubert and G. Müller-Starck

A cyclophilin gene marker confirming geographical differentiation of Norway spruce
populations and indicating viability response on excess soil-born salinity

Molecular Ecology (2003) 12, 3147-3155





































Acknowledgements


I wish to thank Prof. Gerhard Müller-Starck who gave me possibility to work in the DFG
project, commented, edited and added on the chapters of this thesis and the publication in
Molecular Ecology.
I am grateful to Dr. Roland Schubert for his supervision, his help in writting of the article
concerning cyclophilin marker (common with Dr. Ricardo Riegel). I would like to thank
Dr. Roland Schubert and Dr. Ricardo Riegel for the methodological advice in the
laboratory and common discussions.
I wish to say “Thank you very much!” to Eliane Röschter for her great help in the lab and
supporting in the “fight with the complicated computer programs”.
Beatrix Kain and Maria Thole solved all my “bureaucratic” problems. Thanks a lot!
I am thankful to Christiane Bittkau, Sabina La Scala, Rüdiger Baumann, Holger Paetsch,
Cristina Valcu, Sheela Veerasenan for their “every day” help and friendly atmosphere in
the group.
I thank Dr. Ch. Sperisen, Dr. A. Rigling, Dr. T. Skørppa, Dr. I. Shvadchak, Dr. P.
Robakowski, Dr. M Konnert and Dr. F. Bergmann for providing spruce samples.
My mother, my family, my husband Jorge and my daughter Valeria supported me every
day, I thank them.
Contents
Contents

CONTENTS I
ABBREVIATIONS III
FIGURES IV
TABLES VI


1 INTRODUCTION 1

1.1 Genetic variation of forest trees 1
1.2 The role of late Quaternary climatic changes in present
plants distribution in Europe 2
1.3 Picea abies (L.) Karst. 4
1.3.1 History and distribution of Picea abies in Europe 4
1.3.2 Ecological and economical significance of Picea abies 6

1.4 Genetic characterisation of Picea abies 7
1.4.1 Genome 7
1.4.2 Development and application of the genetic markers in Picea abies 9

1.5 EST markers as a new tool in population genetics of forest tree species 15
1.6 Objectives 17

2 MATERIALS AND METHODS 18

2.1 Population sampling 18
2.1.1 Control population of the forest of Kranzberg 18
2.1.2 Populations from European natural range 18
2.2 DNA extraction 20
2.3 Designing and analysing polymorphic EST-PCR marker
2.3.1 Primer construction 21
2.3.2 PCR 21
2.3.3 Electrophoresis in PAG and agarose gels 22
2.3.4 Digestion 23
2.4 Analysing inheritance of EST-PCR marker 24
2.5 DNA sequencing of the polymorphic fragments 24
2.5.1 Cloning 24
2.5.2 Sequencing 26
2.6 Quantifying of genetic variability of Norway spruce 26
Contents

2.6.1 EST markers 27
2.6.2 Estimation of genetic variation 28
2.7 Statistical analysis 31
2.7.1 Test for Hardy-Weinberg proportions 31
2.7.2 Isolation by distance test 31
2.7.3 Neutrality test 32


3 RESULTS 34

3.1 Development of an EST-PCR marker for cyclophilin in Norway spruce 34
3.1.1 cDNA clone encoding cyclophilin, PCR amplification 34
3.1.2 Co-dominant inheritance of an EST marker 35
3.1.3 Sequence data analysis 36
3.2 Monitoring of genetic variation of Norway spruce in Europe 38
3.2.1 Variation within populations 38
3.2.1.1 Allele frequencies at single loci 38
3.2.1.2 Genotype frequencies at single loci 46
3.2.1.3 Heterozygosity 50
3.2.1.4 Diversity 50
3.2.2 Interpopulational variation 53
3.2.2.1 Genetic distance 53
3.2.2.2 Differentiation among populations 54
3.2.3 Isolation by distance test 57
3.2.4 Neutrality Test 59


4 DISCUSSION 60
4.1 Cyclophilin EST marker 60
4.2 Genetic variation of Picea abies in Europe based on EST markers 61
4.3 Indicative potential of the newly developed cyclophilin gene marker
for different environmental impacts on populations of Norway spruce 65

5 CONCLUDING REMARKS 68
6 SUMMARY 69
7 REFERENCES 70
APPENDIX 80


II Abbreviations

Abbreviations

AFLP Amplified fragment length polymorphism
APS Ammoniumpersulfate
BSA Bovine serum albumin
cDNA Complementary (to an RNA) DNA
cpSSR Chloroplast simple sequence repeats
CTAB Cetyl-trimethyl-ammoniumbromide
DNA Deoxyribonucleic acid
dNTP Deoxyribonucleotide-triphosphate
EDTA Ethylene Diamine TetraAcetate
EST Expressed sequence tag
IPTG Isopropylthiogalactoside
MgCl Magnesium chloride 2
mt DNA Mitochondrial DNA
PAG Polyacrilamide gel
PCR Polymerase chain reaction
RAPD Random amplified polymorphic DNA
RFLP Restriction fragment length polymorphism
RNA Ribonucleic acid
SAMPLs Selective amplification of microsatellites polymorphic loci
SCAR Sequence-characterised amplified region
SSCP Single stranded conformational polymorphism
SSR Simple sequence repeat
STS Sequence-tagged-site
TBE Tris-borate EDTA
TE Tris ethylenediaminetetraacetic acid <

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