博士 / 國立臺灣大學 / 生命科學系 / 105 / Reef-building corals for a mutualistic relationship with photosynthetically active dinoflagellates of the genus Symbiodinium that allows them to dominate high light regions of Earth’s tropical seas. Coral reef ecosystems are threatened by excessive coastal development, environmental pollution, and global climate change, and changes in water quality can lead to the disintegration of the coral-dinoflagellate symbiosis. To gain a better understanding of how changes in temperature, in particular, affect such mutualistic associations, next generation sequencing (NGS) was used to profile the meta-transcriptomes (population of all expressed mRNAs) of all “compartments” of the coral “holobiont” (i.e., coral host+Symbiodinium+bacteria) exposed to 1) elevated temperatures over a long-term timescale and 2) variable temperatures over a short-term one. To uncover the gene mRNAs responsive to temperature change, comprehensive analytical platforms were designed for each experiment; both such platforms were made available to the public as interactive websites.
In the first chapter, we review the literature on coral and their symbiotic algae, and discuss how to analyze NGS datasets of multi-compartment organisms (i.e., meta-transcriptomes). In the second chapter, we discuss the various components of the NGS analytical pipeline/platform, as well as the experimental methods. In the next two chapters, we present the data from the two aforementioned experiments with a specific focus on uncovering which cellular pathways of the coral-dinoflagellate endosymbiosis are most affected by elevated and variable temperature exposure.
In the second chapter, we introduce NGS and discuss how to deal with sequencing errors in order to produce the highest quality sequence datasets. We also discuss various algorithms and facets of Trinity, a popular NGS sequence assembly tool and one that was used to assemble the transcriptomes of this dissertation. The assembled contigs were functionally annotated by via alignment to the following three databases: NCBI’s “nr” , 2) KEGG , and 3) Pfam. Finally, we implemented a variety of ways to query the datasets: 1) via keyword search, 2) via BLAST search, 3) via functional enrichment, and 4) via statistical parameter filtering.
In first experiment, the influence of high temperature on the model reef coral Pocillopora damicornis were explored. P. damicornis specimens originally collected from Houbihu (HBH), Nanwan Bay, Taiwan were exposed to either a control (27°C, n=3) or elevated (30°C, n=3) temperature. After 2 and 36 weeks of temperature exposure, samples were sacrificed for RNA extraction followed by NGS-based RNA-Seq. The Symbiodinium populations were relatively more affected than the host corals in which they resided at the mRNA level, and the differentially expressed genes (DEGs) tended to differ between compartments. An interactive website (http://symbiont.iis.sinica.edu.tw/coral_pdltte/) has been designed to serve as a depository for the sequence data set.
In second experiment, the influence of variable temperatures on a coral symbiosis was addressed. Seriatopora hystrix, a temperature-sensitive species, was collected from an upwelling reef in Nanwan Bay, Houbihu, as well as from a non-upwelling site, Houwan. The samples from each region were divided into six tanks and exposed to either a stable temperature (26ºC, n=3) or a variable temperature profile (23-29ºC over a 6-hr cycle, n=3) for one week. After one week of treatment exposure, RNAs were extracted from 12 samples (3/treatment x site of origin group) and sequenced with an Illumina platform. The results revealed that transcriptome varied more across sampling sites than temperature regimes. Also, an interactive website (http://symbiont.iis.sinica.edu.tw/s_hystrix/) has been designed to serve as a depository for the sequence data set.
NGS datasets are immense in size and notoriously difficult to analyze; labs using different pipelines may very well come to entirely different conclusions from another lab handed the same sequence dataset. Such analytical difficulties are compounded in meta-transcriptomic analyses, where mRNA populations are of mixed origin. We therefore believe the analytical platforms and websites developed as part of this dissertation have dramatically advanced the meta-transcriptomics field and may be useful to not only those studying coral-dinoflagellate endosymbioses, but also others interested in profiling the transcriptomes of environmental samples in which RNAs may have emerged from a plethora of different species. Ideally, others will adopt this same pipeline such that meta-transcriptome analyses become more streamlined in the near future, allowing for easier comparison across species, sequencing platforms, and laboratories.
| Identifer | oai:union.ndltd.org:TW/105NTU05525016 |
| Date | January 2017 |
| Creators | Yu-Bin Wang, 王育彬 |
| Contributors | , 于宏燦, 林仲彥 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 179 |
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