Annotation and comparative analysis of fungal genomes: a hitchhiker's guide to genomics

This thesis describes several genome-sequencing projects such as those from the fungi Laccaria bicolor S238N-H82, Glomus intraradices DAOM 197198, Melampsora laricis-populina 98AG31, Puccinia graminis, Pichia pastoris GS115 and Candida bombicola, as well as the one of the haptophyte Emiliania huxleyi CCMP1516. These species are important organisms in many aspects, for instance: L. bicolor and G. intraradices are symbiotic fungi growing associate with trees and present an important ecological niches for promoting tree growth; M. laricis- populina and P. graminis are two devastating fungi threating plants; the tiny yeast P. pastoris is the major protein production platform in the pharmaceutical industry; the biosurfactant production yeast C. bombicola is likely to provide a low ecotoxicity detergent and E. huxleyi places in a unique phylogeny position of chromalveolate and contributes to the global carbon cycle system. The completion of the genome sequence and the subsequent functional studies broaden our understanding of these complex biological systems and promote the species as possible model organisms. However, it is commonly observed that the genome sequencing projects are launched with lots of enthusiasm but often frustratingly difficult to finish. Part of the reason are the ever-increasing expectations regarding quality delivery (both with respect to data and analyses). The Introductory Chapter aims to provide an overview of how best to conduct a genome sequencing project. It explains the importance of understanding the basic biology and genetics of the target organism. It also discusses the latest developments in new (next) generation high throughput sequencing (HTS) technologies, how to handle the data and their applications. The emergence of the new HTS technologies brings the whole biology research into a new frontier. For instance, with the help of the new sequencing technologies, we were able to sequence the genome of our interest, namely Pichia pastoris. This tiny yeast, the analysis of which forms the bulk of this thesis, is an important heterologous production platform because its methanol assimilation properties makes it ideally suitable for large scale industrial production. The unique protein assembly pathway of P. pastoris also attracts much basic research interests. We used the new HTS method to sequence and assemble the GS115 genome into four chromosomes and made it publicly available to the research community (Chapter 2 and Chapter 3). The public release of the GS115 brought broader interests on the comparison of GS115 and its parental strains. By sequencing the parental strain of GS115 with different new sequencing platforms, we identified several point mutations in the coding genes that likely contribute to the higher protein translocation efficiency in GS115. The sequence divergence and copy number variation of rDNA between strains also explains the difference of protein production efficiency (Chapter 4). Before 2008, the Sanger sequencing method was the only technology to obtain high quality complete genomes of eukaryotes. Because of the high cost of the Sanger method, regarding the other genome projects discussed in this thesis, it was necessary to team up with many other partners and to rely on the U.S. Department of Energy Joint Genome Institute (DOE-JGI) and the Broad Institute to generate the genome sequence. The M. larici-populina srain 98AG31 and the Puccinia graminis f. sp. tritici strain CRL 75-36-700-3 are two devastating basidiomycete ‘rusts’ that infect poplar and wheat. Lineage-specific gene family expansions in these two rusts highlight the possible role in their obligate biotrophic life-style. Two large sets of effector-like small-secreted proteins with different pri- mary sequence structures were identified in each organism. The in planta-induced transcriptomic data showed upregulation of these lineage-specific genes and they are likely involved in the establishing of the rust-host interaction. An additional immunolocalization study on M. larici-populina confirmed the accumulation of some candidate effectors in the haustoria and infection hyphae, which is described in Chapter 5

Liquid Density Sensing Using Resonant Flexural Plate Wave Device with Sol-Gel PZT Thin Films

This paper presents the design, fabrication and preliminary experimental results of a flexure plate wave (FPW) resonator using sol-gel derived lead zirconate titanates (PZT) thin films. The resonator adopts a two-port structure with reflecting grates on the composite membrane of PZT and SiNx. The design of the reflecting grate is derived from a SAW resonator model using COM theory to produce a sharp resonant peak. The comparison between the mass and the viscosity effects from the theoretical expression illustrates the applications and the constraints of the proposed device in liquid sensing. Multiple coatings of sol-gel derived PZT films are adopted because of the cost advantage and the high electromechanical coupling effect over other piezoelectric films. The fabrication issues of the proposed material structure are addressed. Theoretical estimations of the mass and the viscosity effects are compared with the experimental results. The resonant frequency has a good linear correlation with the density of low viscosity liquids, which demonstrate the feasibility of the proposed device.Comment: Submitted on behalf of EDA Publishing Association (http://irevues.inist.fr/EDA-Publishing

Fabrication of a microresonator-fiber assembly maintaining a high-quality factor by CO2 laser welding

We demonstrate fabrication of a microtoroid resonator of a high-quality (high-Q) factor using femtosecond laser three-dimensional (3D) micromachining. A fiber taper is reliably assembled to the microtoroid using CO2 laser welding. Specifically, we achieve a high Q-factor of 2.12*10^6 in the microresonator-fiber assembly by optimizing the contact position between the fiber taper and the microtoroid.Comment: 7 pages, 5 figure

Comparative in silico analysis of SSRs in coding regions of high confidence predicted genes in Norway spruce (Picea abies) and Loblolly pine (Pinus taeda)

Background: Microsatellites or simple sequence repeats (SSRs) are DNA sequences consisting of 1-6 bp tandem repeat motifs present in the genome. SSRs are considered to be one of the most powerful tools in genetic studies. We carried out a comparative study of perfect SSR loci belonging to class I (>= 20) and class II (>= 12 and < 20 bp) types located in coding regions of high confidence genes in Picea abies and Pinus taeda. SSRLocator was used to retrieve SSRs from the full length CDS of predicted genes in both species. Results: Trimers were the most abundant motifs in class I followed by hexamers in Picea abies, while trimers and hexamers were equally abundant in Pinus taeda class I SSRs. Hexamers were most frequent within class II SSRs followed by trimers, in both species. Although the frequency of genes containing SSRs was slightly higher in Pinus taeda, SSR counts per Mbp for class I was similar in both species (P-value = 0.22); while for class II SSRs, it was significantly higher in Picea abies (P-value = 0.00009). AT-rich motifs were higher in abundance than the GC-rich motifs, within class II SSRs in both the species (P-values = 10(-9) and 0). With reference to class I SSRs, AT-rich and GC-rich motifs were detected with equal frequency in Pinus taeda (P-value = 0.24); while in Picea abies, GC-rich motifs were detected with higher frequency than the AT-rich motifs (P-value = 0.0005). Conclusions: Our study gives a comparative overview of the genome SSRs composition based on high confidence genes in the two recently sequenced and economically important conifers and, also provides information on functional molecular markers that can be applied in genetic studies in Pinus and Picea species

Experimental Testing and Validation of P-band Bi-static Remote Sensing of Soil Moisture in 137-138MHz Range

Remote sensing using readily available communication signal transmitted by ORBCOMM satellites at very high frequency (VHF) range (137-138MHz) is a promising method for detecting the root zone soil moisture content. The radio wave reflectivity of soil is strongly correlated to soil moisture content. Therefore, if we were able to measure the reflectivity, we might be able to estimate the soil moisture content. In this preliminary study, we analyze direct signal data from the satellites to investigate and verify communication channels in frequency range of interest and their characteristics (bandwidth, pattern, etc.). The analysis of direct signal data is also used for calibrating signal collection systems and compensating for the subtle differences of systems. After comparing the satellite geometry and spectrum from raw signal, we verified that ORBCOMM has 13 channels in our frequency range of interest. It was also verified that among these 13 channels, the channel with center frequency at 137.56MHz is a public channel shared by all satellites and is not suitable for reflectivity computation in that multiple satellites could be in sight by our antenna and the signal reflecting region cannot be determined. In our long duration (~12 hours) analysis, we observed the visible duration and period of the satellites. Conclusively, using ORBCOMM communication signal for sensing the soil moisture is viable. Further study is needed to build up model that relates soil moisture content to reflectivity and a lot of technical issues need to be resolved