Analysis of differential gene expression under water-deficit stress and genetic diversity in bambara groundnut (Vigna subterranea (L.) Verdc.) using novel high-throughput technologies [Elektronische Ressource] / Florian Stadler

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• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2009
• Nombre de lectures: 22
• Langue: Deutsch
• Poids de l'ouvrage: 2 Mo

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TECHNISCHE UNIVERSITÄT MÜNCHEN

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Analysis of differential gene expression under water-deficit stress
and genetic diversity in bambara groundnut
[Vigna subterranea (L.) Verdc.] using novel high-throughput
technologies


Florian Stadler


Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für
Ernährung, Landnutzung und Umwelt der Technischen Universität München zur Erlangung
des akademischen Grades eines

Doktors der Naturwissenschaften

genehmigten Dissertation.


Vorsitzender: Univ.-Prof. Dr. A. Gierl
Prüfer der Dissertation: 1. Univ.-Prof. Dr. G. Wenzel
2. Priv.-Doz. Dr. V. Mohler


Die Dissertation wurde am 23.09.2009 bei der Technischen Universität München eingereicht
und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung
und Umwelt der Technischen Universität München am 23.12.2009 angenommen.
Teilergebnisse dieser Arbeit wurden vorab veröffentlicht:

Publikation:
Mayes, S.*, Basu, S.*, Murchie, E., Roberts, J.A., Azam-Ali, S.N., Stadler, F.*, Mohler, V.,
Wenzel, G., Massawe, F., Kilian, A., Bonin, A., Beena, A., Sheshshayee, M.S. (2009):
BAMLINK – a Cross Disciplinary Programme to Enhance the Role of Bambara Groundnut
(Vigna subterranea L. Verdc.) for Food Security in Africa and India. Acta Horticulturae 806,
137-150.
* Have contributed equally to the data presented in this paper.


Tagungsbeitrag:
Stadler, F., Mohler, V., Kilian, A., Bonin, A., Thümmler, F., Mayes, S., Ros, B., Wenzel, G.
(2007): Applying biotechnology to underutilised crops: initial results from applying Diversity
Arrays Technology (DArT) and Massively Parallel Signature Sequencing to bambara
groundnut. New Approaches to Plant Breeding of Orphan Crops in Africa, 19-21 September
2007, Bern, Switzerland. Abstracts 66.
Table of contents I


1. Introduction ............................................................................................................................ 1
1.1 Bambara groundnut: general information ........................................................................ 1
1.2 Water-deficit stress........................................................................................................... 3
1.2.1 Background ............................................................................................................... 3
1.2.2 The impact of water-deficit on plants ....................................................................... 4
1.2.3 Plant adaptation to water-deficit ............................................................................... 5
1.2.3.1 Late embryogenesis abundant proteins .............................................................. 6
1.2.3.2 Osmolytes and soluble sugars ............................................................................ 6
1.2.3.3 Antioxidants ....................................................................................................... 7
1.2.4 Water deficit stress sensing and signalling ............................................................... 8
1.2.5 Breeding for water-deficit tolerance 10
1.3 The BAMLINK project.................................................................................................. 11
1.3.1 Genetic diversity ..................................................................................................... 12
1.3.2 Gene expression profiling ....................................................................................... 14
1.4 Objectives of the work ................................................................................................... 15
2. Materials and methods.. 17
2.1 DArT .............................................................................................................................. 17
2.1.1 Plant materials......................................................................................................... 17
2.1.2 DNA isolation 17
2.1.3 Marker discovery and scoring................................................................................. 18
2.1.4 Plasmid isolation and sequencing ........................................................................... 20
2.1.5 Statistics and cluster analysis .................................................................................. 21
2.2 Gene expression under water-deficit stress.................................................................... 21
2.2.1 Water-deficit stress experiments ............................................................................. 21
2.2.1.1 Plant materials.................................................................................................. 21
2.2.1.2 Growing conditions .......................................................................................... 23
2.2.1.3 Experimental set-up and sampling ................................................................... 23
2.2.1.4 Physiological measurements ............................................................................ 24
2.2.2 RNA isolation.......................................................................................................... 25
2.2.3 cDNA libraries and high-throughput pyrosequencing ............................................ 26
2.2.4 MPSS data analysis ................................................................................................. 27
2.2.5 Selection of sequences for microarray analysis ...................................................... 28
2.2.6 Oligonucleotide and microarray design .................................................................. 28
2.2.7 cDNA synthesis and hybridisation.......................................................................... 29
2.2.8 Microarray scanning and data analysis ................................................................... 30
3. Results .................................................................................................................................. 31
3.1 Genetic diversity.. 31
3.1.1 Initial discovery array.............................................................................................. 31
3.1.2 Development of full-size array................................................................................ 33 II Table of contents

3.1.3 Genotyping and cluster analysis.............................................................................. 34
3.1.4 Large-scale genotyping ........................................................................................... 37
3.1.5 Intra-landrace diversity and ‘exotic’ germplasm .................................................... 38
3.2 Gene expression under water-deficit stress.................................................................... 41
3.2.1 MPSS expression profiling...................................................................................... 41
3.2.1.1 Data assembly and analysis.............................................................................. 41
3.2.1.2 Global water-deficit response........................................................................... 43
3.2.1.3 Genotypic differences 47
3.2.2 Microarrays ............................................................................................................. 51
3.2.2.1 Signal linearity ................................................................................................. 51
3.2.2.2 Validation of MPSS ......................................................................................... 51
3.2.3 Time course experiment .......................................................................................... 53
3.2.3.1 Physiological studies........................................................................................ 53
3.2.3.2 Expression kinetics of selected genes .............................................................. 56
3.2.3.2.1 Hierarchical cluster analysis...................................................................... 56
3.2.3.2.2 k-Means cluster analysis ........................................................................... 58
3.2.3.3 Within-time point analysis ............................................................................... 63
3.2.3.3.1 Differential gene expression between treatments...................................... 63
3.2.3.3.2 Landrace-specific differences ................................................................... 70
4. Discussion ............................................................................................................................ 73
4.1 Genetic diversity ............................................................................................................ 73
4.1.1 Experimental approach............................................................................................ 73
4.1.2 Cluster analysis ....................................................................................................... 77
4.1.3 Intra-landrace diversity and “exotic” germplasm.................................................... 79
4.2 Gene expression under wate