Isolation and characterization of nuclear and mitochondrial genetic markers for population studies of Ucides cordatus cordatus (Decapoda: Brachyura) [Elektronische Ressource] / vorgelegt von Marco Ewald

Besitzer

Sujets

• Biologie

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• Publié par: universitat_bremen
• Publié le: 01 janvier 2006
• Nombre de lectures: 33
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

Isolation and Characterization of Nuclear and
Mitochondrial Genetic Markers for Population
Studies of Ucides cordatus cordatus (Decapoda:
Brachyura)
Dissertation
zur Erlangung des Grades eines
Doktors der Naturwissenschaften (Dr. rer. nat.)
vorgelegt von
Marco Ewald
angefertigt am
Zentrum für Umweltforschung und Technologie (UFT)
Abteilung Molekulare Genetik und Biotechnologie
innerhalb des Fachbereichs 2 der Universität Bremen
Bremen 2006
iDrawing on cover: The uçá, Ucides cordatus cordatus (Linnaeus 1763) as
depicted in Zacharias Wagner's "Thier Buch," c. 1640. Reproduced from
Dutch Brazil (Rio de Janeiro: Editora Index, 1997), vol. 2, plate 25.
iiPrincipal supervisor: Prof. Dr. Dietmar Blohm, UFT at the University of
Bremen, Germany
Co-supervisor: Prof. Dr. Horacio Schneider, at the Laboratório de Genética e
Biologia Molecular- Nucleo de Estudos Costeiros, UFPA, Federal University of
Para Bragança, Para, Brazil
iiiContents
ACKNOWLEDGMENTS………………………………………………………. vi
SUMMARY........................................…………………................................ viii
ZUSAMMENFASSUNG............................................................................. xi
LIST OF TABLES AND FIGURES…………………………………………… xv
1 Introduction…………………………………………………………… 1
…
1.1 1The Mangrove Ecosystem……………………………………………..
1.2 MADAM Project (Mangrove Dynamics and Management)………... 2
1.3 The semi terrestric crab Ucides 4
cordatus…………………………….
1.3.1 Ecological Role…………………………………………………………. 4
1.32 Systematic of Ucides cordatus……………………………………….. X
Xxx Economical Role...............................................................………… 6
1.4 9Objectives and
Studies…………………………………………………
1.5 DNA Polymorphism… 10
1.5.1 13Microsatellites…………………………………………………………..
1.5.2 Stutterbands……………………………………………………………. 15
1.5.3 Mitochondrial DNA…………………………………………………….. 16
1.5.4 17Cytochrome Oxidase Subunit I (COI) Coding DNA…………………
1.5.5 Mitochondrial Pseudogenes………………………………………...... 19
2 Material and Methods………………………………………………….. 22
2.1 24Probe Sampling…………………………………………………………
2.2 Isolation and Characterisation of Microsatellites…………………… 24
2.2.1 DNA Extraction………………………………………………………… 24
2.2.2 Microsatellite Screening and Amplification…………………………. 27
2.2.3 27DNA Sequencing and Primer Design………………………………..
2.2.4 Analyzing of the Allele Frequencies………… 27
2.2.5 Genotyping and population analyses of Ucides cordatus…………. 28
2.3 28COI Approach.............................................................……………..
iv2.3.1 30DNA Extraction and Sequencing……………………………………..
2.3.2 Phylogenetic and Population Variability Analyses Using COI
Data……………………………………………………………………… 32
3 32Results....................................................................................…….
.
3.1 Isolation of Microsatellite Sequences in Ucides cordatus…………. 32
Characterisation of Selected Microsatellite Loci……………………………3.2 36
3.3 Diversity of Selected Microsatellite Loci……………………………... 37
3.4 Characterisation of the Partial COI Coding Sequence…………….. 42
3.5 Diversity of the COI Coding DNA…………………………………….. 45
3.6 49Demographic Investigation Using the COI Marker………………….
4 Discussion………………………………………………………………. 49
4.1 Isolation and Characterisation of Microsatellite Loci……………….. 53
4.2 55Experimental Uncertainties in Using Selected Microsatellite
Loci……………………………………………………………………….
4.3 Microsatellite Diversity in Ucides Populations………………………. 56
4.4 Mitochondrial COI Gene in Population Genetics of U. 56
cordatus………………………………………………………………….
4.4.1Diversity of the COI 57
Gene……………………………………………...
4.4.2 Population Dynamics of Ucides cordatus…………………………… 60
4.4.3 Putative Pseudogenes in Ucides cordatus…………………………. 61
4.5 Comparison of Population Divergence from Mitochondrial COI
62DNA and Nuclear Microsatellite Loci…………………………………
4.6 Marine dispersal of Ucides cordatus...............................………… 65
5 References…………………………………………………………….. 65
vAcknowledgements
I would like to thank Prof. Dr. Dietmar Blohm for having given me the
opportunity to conduct this thesis, for his support and very helpful advices.
Many thanks go to Prof. Dr. Horatio Schneider at the Laboratório de Genética
e Biologia Molecular- Nucleo de Estudos Costeiros, UFPA, Federal University
of Para Bragança, Brazil for his unresting help, support and fruitful
discussions during my stay in Brazil. Thanks a lot for accepting to be my
second superviser as well as offering the possibility for finishing my PhD in his
group in Brazil.
Many thanks to Prof. Dr. Ulli Saint-Paul for iniciating the scientific question
and for imbedding the study into the MADAM project.
My special thanks go to Prof. Dr. Iracilda Sampaio for supporting my work
during the last months in Brazil, for helping on the practical as well as
theoretical part of this study and supporting to establish myself in an unknown
country.
Many thank to the members of the group of Biotechnology and Molecular
Genetics at the University of Bremen and the members of the Laboratório de
Genética e Biologia Molecular- Nucleo de Estudos Costeiros in Bragança for
a very open and helpful atmosphere and several helpful comments, too.
Substitutional for all nice collegues I would like to thank Dr. Sascha Todt and
viTawfiq Froukh for discussions and friendship in Bremen, either Dr. Marcello
Vallinoto for supporting my statistical analyses and several proposals to
improve the population genetic work in Brazil.
Further many thanks to my mother Elise and several friends substitutional
Moirah, Stumpi and Christiane who did accompany my path of live over years.
Without you realising my life´s dream would be much more difficult.
viiSummary
The mangrove ecosystem has fundamental influences on storm protection of the coast,
erosion control, and wastewater cleanup. In Brazil, one of the key species of this
ecosystem is Ucides cordatus. In the mangrove forests it accounts for up to 84% of
the epibenthic biomass and is of highest importance by fuelling the detritus based
food web by accelerating the disruption of organic matter falling from the trees.
Further, there is a high significance of crustacean fisheries in general and of U.
cordatus especially as a major financial income for rural populations in coastal
regions. In parts of the mangrove forests the same amount of this resource produced
each year is harvested. At the same time, U. cordatus is a slow growing species and
requires 6 to 11 years to reach market size of about 7 cm and needs up to 3 years to be
able to reproduce. Due to this economically and ecologically importance of U.
cordatus this species should be included into a coastal management plan to protect the
mangrove forests and to manage the edible resources like U. cordatus. Therefore
information about distribution and population dynamics is necessary to adequate
elements for sustainable exploitation and managing. Especially because the larvae
dispersal and the remigration mechanism to the mangrove forests is not clear yet and
to what extent the population of U. cordatus in the mangroves could originate from
other regions. The objective of this study is to develop a DNA based marker system to
analyze the following questions. Are there polymorphisms between morphologically
equal individuals in selected potentially variable DNA markers and are they available
to measure gene flow, genetic population structure and exchanges of individuals
between separated geographically locations? Further it should be examined if there
are any indications that the species U. cordatus is still over fished.
viiiAs nuclear genetic marker in this study 110 microsatellite loci were successfully
isolated from different enriched partial libraries of U. cordatus and with 5 of them
population analyzes were done using 135 individuals from Bragança and Paraná. The
microsatellite markers developed here are the first for Ucides and show high
polymorphism with 16 to 47 different alleles, respectively and values of observed
heterozygosity from 0.23 to 0.88 with values of expected heterozygosity from 0.87 to
0.98. Significant deviations from Hardy Weinberg equilibrium (HWE) were detected
for all tests at each locus in the population of Bragança and Paraná with a deficit of
heterozygosity of -0.08 to -0.72 and zero allele estimation with an amount of 0.043 to
0.417. F-statistics detects a low but highly significant variability between the
populations of Bragança and Paraná with Fst values of 0.011 to 0.048 in 4 of 5 loci
and therefore restricted gene flow supported by P values lowers than 0.01. Because of
the possible error rate in genotyping through stutter bands and suggested high
amounts of zero alleles the weak population structure found in U. cordatus was
confirmed using an additional different marker. A part of the mitochondrial gene
coding for the Cytochrome oxidase subunit I (COI), was amplified and 600 bp were
sequenced using 223 individuals pooled from the regions “Upper Amazon”, “Lower
Amazon” and “South Brazil”. A high amount of polymorphism was found with 132
different haplotypes resulting in a diversity of 0.97 within the