Exploring Genome Structure and Gene Regulation Related to Virulence in Fungal Phytopathogens Using Next Generation Sequencing Techniques
2013
Over the last decade, a technological revolution has
provided enormous advances in the knowledge of complex biological
processes largely enabled by the development of next-generation
sequencing (NGS) techniques. Applications of NGS include studies of
entire genomes, characterization of the entire transcriptome
(RNA-Seq), and detection of protein-DNA binding sites (ChIP-Seq).
The cost of sequencing a fungal genome, for example, has decreased
from more than one hundred thousand dollars to currently only three
thousand dollars. With the development of the applications and the
affordable cost, NGS is changing the way biologists designing and
carrying out research. This dissertation describes developed
analysis pipelines of NGS data in the field of fungal
phytopathogens using four different projects as examples. In a
genome comparison project, sequenced short reads are de novo
assembled to form a genome draft, then gene models are predicted
either ab initial or assisted by RNA-Seq, followed by the
comparison between genomes at different resolutions such as the
nucleotide level and the genome structural level. Two chapters in
this dissertation serve as examples of our genome comparison
pipeline put to work to address biological questions. In Chapter 2,
the Alternaria arborescens sequences of the unique conditionally
dispensable chromosome (CDC) were separated from essential
chromosomes (EC) using a novel bioinformatics approach. A pair-wise
comparison between the CDC and ECs showed that CDC sequences had
significant variation and that it may have been originally acquired
through a horizontal gene transfer event. In Chapter 4, seven field
isolates of Magnaporthe oryzae were sequenced and their genome
content compared. Over 10,000 SNP and Indel locations were
identified as well as genes under strong positive selection, which
are considered potential virulence related genes. While in a
RNA-Seq analysis pipeline, sequence reads are first assembled de
novo or mapped to a reference genome, and the expression level for
individual genes in each sequencing library is calculated to
identify differentially expressed genes. Chapter 3 describes a
RNA-Seq analysis project, in which the transcriptome profile in the
dollar spot pathosystem has been sequenced using a combination of
Illumina and Roche 454 NGS technologies. A large number of genes
were found up-regulated during the interaction between Sclerotinia
homoeocarpa and Agrostis stolonifera with some having annotations
suggesting their roles in virulence related processes. With regard
to protein-DNA binding, reads from ChIP-Seq experiment are mapped
to the reference genome and the “peak regions” of mapped reads are
identified as candidate binding regions, within which binding
motifs are predicted. Chapter 5 describes the identifications of
the binding sites and motifs of the M. oryzae transcription factor
MoCRZ1 using a combination of ChIP-chip and microarray data, and
then the prediction accuracy is improved by a novel approach
utilizing the spatial distribution pattern of the…
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