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Language(s):
eng - English
Dates:
2017-112017-11
Publication Status:
Issued
Pages:
72
Publishing info:
Kiel
Table of Contents:
Table of Contents
1. Introduction ........................................................................................................................ 1
1.1 Methods for exploring microbial diversity.................................................................. 3
1.1.1 Culture Methods................................................................................................... 3
1.1.2 Culture-independent methods .............................................................................. 3
1.1.3 High throughput Metabarcoding .......................................................................... 5
1.2 Challenges in Phyllosphere endophytic microbial diversity research ......................... 7
1.2.1 Highly similar host cellular DNA sequences ....................................................... 7
1.3 Previous research in phyllosphere microbial diversity ............................................... 9
1.4 Thesis aim ................................................................................................................. 10
2. Materials and Methods ..................................................................................................... 12
1.5 Study design .............................................................................................................. 12
1.6 Sampling of wheat leaves .......................................................................................... 12
1.7 Leaf surface sterilization ........................................................................................... 12
1.7.1 Microbial growth assay ...................................................................................... 14
1.7.2 PCR assay .......................................................................................................... 14
1.8 Methods for DNA extraction .................................................................................... 14
1.8.1 Qiagen kit method .............................................................................................. 14
1.8.2 Modified CTAB (Cetyltrimethylammonium bromide) method ........................ 15
1.8.3 Phenol-Chloroform based DNA extraction protocol ......................................... 15
1.9 Design of blocking primers ....................................................................................... 16
1.10 Clone library method ................................................................................................. 17
1.11 Next-generation sequencing ...................................................................................... 17
1.11.1 Amplicon library preparation ............................................................................. 18
1.11.2 Amplicon Sequencing ........................................................................................ 18
1.12 Data Analyzes ........................................................................................................... 19
3. Results .............................................................................................................................. 21
1.13 Optimization of surface sterilization ......................................................................... 21
1.13.1 Results from Microbial growth assay ................................................................ 21
1.13.2 Results from PCR assay ..................................................................................... 21
1.14 Comparison of DNA extraction methodologies ........................................................ 22
1.14.1 Results from Qiagen kit method ........................................................................ 23
1.14.2 Results from modified CTAB method ............................................................... 23
1.14.3 Results from Phenol-Chloroform based DNA extraction method ..................... 23
1.15 Design of blocking primers ....................................................................................... 24
1.16 Preliminary screening of endophytes with Clone library method ............................. 27
1.17 Library preparation and Amplicon sequencing ......................................................... 28
1.18 Data pre-processing and OTU picking ...................................................................... 29
1.19 Structure of bacterial and fungal endophyte communities ........................................ 30
1.19.1 Taxonomy, relative abundances and taxa summary .......................................... 30
1.20 Diversity analyzes ..................................................................................................... 32
1.20.1 Alpha Diversity .................................................................................................. 32
1.20.2 Beta Diversity .................................................................................................... 34
1.20.3 PCoA and NMDS .............................................................................................. 35
1.21 Metabolic pathways prediction in bacterial communities ......................................... 36
1.22 Identification of differentially abundant taxa ............................................................ 37
4. Discussion ........................................................................................................................ 38
1.23 Highly efficient protocol for metaprofiling of wheat phyllosphere endophyte
communities ......................................................................................................................... 38
1.24 The timing of sterilization and freezing process doesn’t have significant effect on
the microbial diversity ......................................................................................................... 42
1.25 Structure of bacterial and fungal communities and the impact of field site on the
microbial diversity ............................................................................................................... 43
1.26 Conclusions and future perspectives ......................................................................... 46
5. Acknowledgement ........................................................................................................... 48
6. References ........................................................................................................................ 49
Rev. Type:
-
Identifiers:
Other: Dipl/12918
Degree:
Master
