National Center for Genome Analysis Program Year 3 Report – September 15, 2013 – September 14, 2014

On September 15, 2011, Indiana University (IU) received three years of support to establish the National Center for Genome Analysis Support (NCGAS). This technical report describes the activities of the third 12 months of NCGASThe facilities supported by the Research Technologies division at Indiana University are supported by a number of grants. The authors would like to acknowledge that although the National Center for Genome Analysis Support is funded by NSF 1062432, our work would not be possible without the generous support of the following awards received by our parent organization, the Pervasive Technology Institute at Indiana University. • The Indiana University Pervasive Technology Institute was supported in part by two grants from the Lilly Endowment, Inc. • NCGAS has also been supported directly by the Indiana METACyt Initiative. The Indiana METACyt Initiative of Indiana University is supported in part by the Lilly Endowment, Inc. • This material is based in part upon work supported by the National Science Foundation under Grant No. CNS-0521433. Any opinions, findings and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation (NSF)

Genomic and Transcriptomic Analysis of the Anerobic Fungus Orpinomyces Strain C1a, a Versatile Biodegrader of Plant Biomass

The anaerobic fungi represent a basal fungal lineage, members of which reside in the rumen and alimentary tract of herbivores. Due to their reported capacity to degrade plant materials, the anaerobic fungi have recently been touted as promising agents for biofuel production. In the first part of this thesis, I present the first reported genomic analysis of a member of the anaerobic gut fungi, Orpinomyces sp. strain C1A. The genome of strain C1A was sequenced using a combination of Illumina and PacBio SMRT technologies. The large genome (100.95 Mb, 16,347 genes) displayed extremely low G+C content (17.0%), large non-coding intergenic regions (73.1%), a proliferation of microsatellite repeats (4.9%), and multiple gene duplications. Comparative genomic analysis identified multiple genes and pathways that are absent in Dikarya genomes but present in basal fungal lineages and/or non-fungal Opisthokonts. Analysis of the lignocellulolytic machinery in the C1A genome revealed an extremely rich repertoire, with evidence of horizontal gene acquisition from multiple bacterial lineages. Experimental analysis indicated that strain C1A is a remarkable biomass degrader, capable of simultaneous saccharification and fermentation of the cellulosic and hemicellulosic fractions in multiple untreated grasses and crop residues examined, with the process significantly enhanced by mild pretreatments.In the second part of my thesis, I analyzed the transcriptomic profiles of C1A when grown on four different types of lignocellulosic biomass (alfalfa, energy cane, corn stover, and sorghum) versus a soluble sugar monomer (glucose).The thesis hence represents the first in-depth evaluation of the genome and transcriptome of a member of this poorly studied group of fungi. Collectively, my work has revealed multiple novel insights into the metabolic capabilities, cell biology, and genomic architecture of anaerobic fungi such as the presence of unique pathways and processes not encountered in higher fungi, genomic features shaped by its unique evolutionary trajectory, extensive lignocellulolytic gene repertoire, and regulatory mechanisms employed to achieve fast and efficient biomass degradation within the herbivore gut.Microbiology, Cell, & Molecular Biolog

Black perithecial pigmentation in <i>Fusarium </i>species is due to the accumulation of 5-deoxybostrycoidin-based melanin

Biosynthesis of the black perithecial pigment in the filamentous fungus Fusarium graminearum is dependent on the polyketide synthase PGL1 (oPKS3). A seven-membered PGL1 gene cluster was identified by over-expression of the cluster specific transcription factor pglR. Targeted gene replacement showed that PGL1, pglJ, pglM and pglV were essential for the production of the perithecial pigment. Over-expression of PGL1 resulted in the production of 6-O-demethyl-5-deoxybostrycoidin (1), 5-deoxybostrycoidin (2), and three novel compounds 5-deoxybostrycoidin anthrone (3), 6-O-demethyl-5-deoxybostrycoidin anthrone (4) and purpurfusarin (5). The novel dimeric bostrycoidin purpurfusarin (5) was found to inhibit the growth of Candida albicans with an IC(50) of 8.0 +/− 1.9 μM. The results show that Fusarium species with black perithecia have a previously undescribed form of 5-deoxybostrycoidin based melanin in their fruiting bodies

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Identification and characterisation of genes controlling the resistance response to ascochyta blight (Ascochyta rabiei (Pass.) Labrousse) in chickpea (Cicer arietinum L.)

Ascochyta blight, caused by Ascochyta rabiei (Pass.) Labrousse, is one of the most destructive diseases of chickpea (Cicer arietinum L.) worldwide. Despite the existence of highly resistant uncultivated genotypes, attempts to develop cultivars with a high level of durable resistance have been unsuccessful. This study investigated the chickpea defence response to A. rabiei using a functional genomics approach, which has the capacity to improve the overall understanding of the coordinated defence response at a molecular level. An existing cDNA library was used to generate a resource of Expressed Sequence Tags (ESTs) that, after clustering, comprised 516 unigenes. The unigenes were functionally annotated resulting in the identification of 20 specific defence-related unigenes, as well as numerous transcripts with possible involvement in the coordination of defence responses. To explore the expression patterns of the defence-related unigenes in an A. rabiei resistant and susceptible genotype, the unigenes were employed as probes in microarrays. Resulting expression data was analysed to identify differentially expressed unigenes over a time-course after infection. Comparison of the expression profiles from the resistant and susceptible genotype identified three putative genes that were exclusively up-regulated in the resistant genotype, thus may be involved in an effective defence response. Considering that a defence response can involve hundreds of genes, the entire set of chickpea unigenes were used to construct large-scale microarrays. To supplement the chickpea probes, 156 putative defence-related grasspea (Lathyrus sativus L.) ESTs and 41 lentil (Lens culinaris Med.) Resistance Gene Analogs (RGAs) were also included. Expression profiles for three chickpeas and one wild relative were generated over a time course. 97 differentially expressed ESTs were identified using a robust experimental system that included confirmation by quantitative RT-PCR. The results indicated that genes involved in the active defence response were similar to those governed by R-gene mediated resistance, including the production of reactive oxygen species and the hypersensitive response, down-regulation of &#039;housekeeping&#039; gene expression, and expression of pathogenesis-related proteins. The comparison between resistant and susceptible genotypes identified certain gene expression &#039;signatures&#039; that may be predictiv e of resistance. To further characterise the regulation of potential defence-related genes, the microarray was used to study expression profiles of the three chickpea genotypes (excluding the wild relative) after treatment with the defence signalling compounds, ethylene (E), salicylic acid (SA), and jasmonate (JA). 425 ESTs were differentially expressed, and comparison between genotypes revealed the presence of a wider range of inducible defence responses in resistant genotypes. Linking the results with the previous microarray results indicated the presence of other pathogen-specific signalling mechanisms in addition to E, SA and JA. The lower arsenal of defence-related gene expression observed in the susceptible genotype may be a result of &#039;breaks&#039; in the pathways of defence-related gene activation. To draw together the findings of all experiments, a model was constructed for a hypothetical mechanism of chickpea resistance to A. rabiei. The model was synthesised based on the evidence gathered in this study and previously documented defence mechanisms in chickpea, and identified signal transduction as a key to resistance

Large-scale sequencing and assembly of cereal genomes using blacklight

Wheat, corn, and rice provide 60 percent of the world's food intake every day, and just 15 plant species make up 90 percent of the world's food intake. As such there is tremendous agricultural and scientific interest to sequence and study plant genomes, especially to develop a reference sequence to direct plant breeding or to identify functional elements. DNA sequencing technologies can now generate sequence data for large genomes at low cost, however, it remains a substantial computational challenge to assemble the short sequencing reads into their complete genome sequences. Even one of the simpler ancestral species of wheat, Aegilops tauschii, has a genome size of 4.36 gigabasepairs (Gbp), nearly fifty percent larger than the human genome. Assembling a genome this size requires computational resources, especially RAM to store the large assembly graph, out of reach for most institutions. In this paper, we describe a collaborative effort between Cold Spring Harbor Laboratory and the Pittsburgh Supercomputing Center to assemble large, complex cereal genomes starting with Ae. tauschii, using the XSEDE shared memory supercomputer Blacklight. We expect these experiences using Blacklight to provide a case study and computational protocol for other genomics communities to leverage this or similar resources for assembly of other significant genomes of interest. Copyright 2014 ACM

Metabolic changes in Arabidopsis thaliana plants overexperssing chalcone synthase

The study has shown that it is possible to introduce the heterologous CHS gene in Arabidopsis thaliana and common multicopies of transgenes containing plants were obtained. Analysis of the change in metabolome of CHS transgenic plants, high expression transgenic lines can be identified by markers such as flavonoids and phenylpropanoids. It is also clear that UV-A/blue light stress does not further increase the levels of these marker compounds in CHS transgenic Arabidopsis plants, whereas in wild type plants such a treatment results in increased levels of these compounds, in fact similar to that in the transgenic plants. There are certain physiological limitations in the accumulation of certain products. This thesis starts with a review of the function of CHS in plants and especially in plant resistance (Chapter 2). Chapter 3 deals with the work on Agrobacterium-mediated transformation of heterologous chalcone synthase in Arabidopsis thaliana Col. 0. The effect of overexpression of CHS on the transcriptional level is discribed in this chapter. The activity of the CHS enzyme in the transgenic plants is reported in Chapter 4. In Chapter 5 metabolic profiling of Arabidopsis thaliana using nuclear magnetic resonance spectroscopy (NMR) is described. In this chapter the primary and secondary metabolites of Arabidopsis thaliana Col. 0 which can be detected by NMR are reported. Chapter 6 reports the metabolic profiling of CHS transgenic Arabidopsis. Metabolomic changes upon UV-A/blue light treatment of Arabidopsis thaliana were investigated (Chapter 7). Chapter 8 deals with the study of the effect of the non-pesticide chemical, Benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester (BTH) on the Arabidopsis metabolome. Finally, the general summary and discussion of thesis are given in Chapter 9.LEI Universiteit LeidenVietnamese governmentMetabolemic