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

A High Quality Genome for Mus spicilegus, a Close Relative of House Mice with Unique Social and Ecological Adaptations

Genomic data for the closest relatives of house mice (Mus musculus species complex) are surprisingly limited. Here, we present the first complete genome for a behaviorally and ecologically unique member of the sister clade to house mice, the mound-building mouse, Mus spicilegus. Using read cloud sequencing and de novo assembly we produced a 2.50 Gbp genome with a scaffold N50 of 2.27 Mbp. We constructed >25 000 gene models, of which the majority had high homology to other Mus species. To evaluate the utility of the M. spicilegus genome for behavioral and ecological genomics, we extracted 196 vomeronasal receptor (VR) sequences from our genome and analyzed phylogenetic relationships between M. spicilegus VRs and orthologs from M. musculus and the Algerian mouse, M. spretus. While most M. spicilegus VRs clustered with orthologs in M. musculus and M. spretus, 10 VRs with evidence of rapid divergence in M. spicilegus are strong candidate modulators of species-specific chemical communication. A high quality assembly and genome for M. spicilegus will help to resolve discordant ancestry patterns in house mouse genomes, and will provide an essential foundation for genetic dissection of phenotypes that distinguish commensal from non-commensal species, and the social and ecological characteristics that make M. spicilegus unique

Gene-rich X chromosomes implicate intragenomic conflict in the evolution of bizarre genetic systems

Haplodiploidy and paternal genome elimination (HD/PGE) are common in invertebrates, having evolved at least two dozen times, all from male heterogamety (i.e., systems with X chromosomes). However, why X chromosomes are important for the evolution of HD/PGE remains debated. The Haploid Viability Hypothesis posits that X-linked genes promote the evolution of male haploidy by facilitating purging recessive deleterious mutations. The Intragenomic Conflict Hypothesis holds that conflict between genes drives genetic system turnover; under this model, X-linked genes could promote the evolution of male haploidy due to conflicts with autosomes over sex ratios and genetic transmission. We studied lineages where we can distinguish these hypotheses: species with germline PGE that retain an XX/X0 sex determination system (gPGE+X). Because evolving PGE in these cases involves changes in transmission without increases in male hemizygosity, a high degree of X linkage in these systems is predicted by the Intragenomic Conflict Hypothesis but not the Haploid Viability Hypothesis. To quantify the degree of X linkage, we sequenced and compared 7 gPGE+X species’ genomes with 11 related species with typical XX/XY or XX/X0 genetic systems, representing three transitions to gPGE. We find highly increased X linkage in both modern and ancestral genomes of gPGE+X species compared to non-gPGE relatives and recover a significant positive correlation between percent X linkage and the evolution of gPGE. These empirical results substantiate longstanding proposals for a role for intragenomic conflict in the evolution of genetic systems such as HD/PGE

Sex Chromosome Transformation and the Origin of a Male-Specific X Chromosome in the Creeping Vole.

The mammalian sex chromosome system (XX female/XY male) is ancient and highly conserved. The sex chromosome karyotype of the creeping vole (Microtus oregoni) represents a long-standing anomaly, with an X chromosome that is unpaired in females (X0) and exclusively maternally transmitted. We produced a highly contiguous male genome assembly, together with short-read genomes and transcriptomes for both sexes. We show that M. oregoni has lost an independently segregating Y chromosome and that the male-specific sex chromosome is a second X chromosome that is largely homologous to the maternally transmitted X. Both maternally inherited and male-specific sex chromosomes carry fragments of the ancestral Y chromosome. Consequences of this recently transformed sex chromosome system include Y-like degeneration and gene amplification on the male-specific X, expression of ancestral Y-linked genes in females, and X inactivation of the male-specific chromosome in male somatic cells. The genome of M. oregoni elucidates the processes that shape the gene content and dosage of mammalian sex chromosomes and exemplifies a rare case of plasticity in an ancient sex chromosome system

De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis

De novo assembly of RNA-seq data enables researchers to study transcriptomes without the need for a genome sequence; this approach can be usefully applied, for instance, in research on 'non-model organisms' of ecological and evolutionary importance, cancer samples or the microbiome. In this protocol we describe the use of the Trinity platform for de novo transcriptome assembly from RNA-seq data in non-model organisms. We also present Trinity-supported companion utilities for downstream applications, including RSEM for transcript abundance estimation, R/Bioconductor packages for identifying differentially expressed transcripts across samples and approaches to identify protein-coding genes. In the procedure, we provide a workflow for genome-independent transcriptome analysis leveraging the Trinity platform. The software, documentation and demonstrations are freely available from http://trinityrnaseq.sourceforge.net. The run time of this protocol is highly dependent on the size and complexity of data to be analyzed. The example data set analyzed in the procedure detailed herein can be processed in less than 5 h.Howard Hughes Medical InstituteNational Institutes of Health (U.S.). Pioneer AwardNational Human Genome Research Institute (U.S.) (Center for Excellence in Genome Science Grant 5P50HG006193-02)Klarman Cell Observator