October 23, 2019  |  

Development of a Novel Reference Transcriptome for Scleractinian Coral Porites lutea Using Single-Molecule Long-Read Isoform Sequencing (Iso-Seq)

Elevation in seawater temperature associated with global climate change has caused coral bleaching problems and posed a significant threat to coral health and survival worldwide. Several studies have explored the effects of thermal stress on changes in gene expression levels of both coral hosts and their algal endosymbionts and provided evidences suggesting that corals could acclimatize to environmental stressors through differential regulation of their gene expression (Desalvo et al., 2008, 2010; Császár et al., 2009; Rodriguez-Lanetty et al., 2009; Polato et al., 2010; Meyer et al., 2011; Kenkel et al., 2013). Such information is crucial for understanding the adaptive capacity of the coral holobionts (Hughes et al., 2003). The availability of transcriptome data from a number of coral species and their associated Symbiodinium allows us to probe the molecular stress response of the organisms to heat stress (Traylor-Knowles et al., 2011; Moya et al., 2012; Kenkel et al., 2013; Shinzato et al., 2014; Kitchen et al., 2015; Anderson et al., 2016; Davies et al., 2016). Here, we report the first reference transcriptome for a scleractinian coral Porites lutea, one of the dominant reef-builders in the Indo-West Pacific (Yeemin et al., 2009). We applied both short-read Ion S5 RNA sequencing and long-read Pacific Biosciences (PacBio) isoform sequencing (Iso-seq) to generate transcriptome sequences of P. lutea under normal and heat stress conditions. The key advantage of PacBio’s Iso-seq technology lies within its ability to capture full-length mRNA sequences. These full-length transcripts enable the identification of novel genes/isoforms and the detection of alternative splice variants, which have been shown to be overrepresented in stress responses (Iida et al., 2004; Reddy et al., 2013; Liu and Guo, 2017). We envision that this reference transcriptome will provide a coral research community a valuable resource for investigating changes in gene expression under various biotic/abiotic stress conditions.

September 22, 2019  |  

Uncovering full-length transcript isoforms of sugarcane cultivar Khon Kaen 3 using single-molecule long-read sequencing.

Sugarcane is an important global food crop and energy resource. To facilitate the sugarcane improvement program, genome and gene information are important for studying traits at the molecular level. Most currently available transcriptome data for sugarcane were generated using second-generation sequencing platforms, which provide short reads. The de novo assembled transcripts from these data are limited in length, and hence may be incomplete and inaccurate, especially for long RNAs.We generated a transcriptome dataset of leaf tissue from a commercial Thai sugarcane cultivar Khon Kaen 3 (KK3) using PacBio RS II single-molecule long-read sequencing by the Iso-Seq method. Short-read RNA-Seq data were generated from the same RNA sample using the Ion Proton platform for reducing base calling errors.A total of 119,339 error-corrected transcripts were generated with the N50 length of 3,611 bp, which is on average longer than any previously reported sugarcane transcriptome dataset. 110,253 sequences (92.4%) contain an open reading frame (ORF) of at least 300 bp long with ORF N50 of 1,416 bp. The mean lengths of 5′ and 3′ untranslated regions in 73,795 sequences with complete ORFs are 1,249 and 1,187 bp, respectively. 4,774 transcripts are putatively novel full-length transcripts which do not match with a previous Iso-Seq study of sugarcane. We annotated the functions of 68,962 putative full-length transcripts with at least 90% coverage when compared with homologous protein coding sequences in other plants.The new catalog of transcripts will be useful for genome annotation, identification of splicing variants, SNP identification, and other research pertaining to the sugarcane improvement program. The putatively novel transcripts suggest unique features of KK3, although more data from different tissues and stages of development are needed to establish a reference transcriptome of this cultivar.

September 22, 2019  |  

Genome characterization of oleaginous Aspergillus oryzae BCC7051: A potential fungal-based platform for lipid production.

The selected robust fungus, Aspergillus oryzae strain BCC7051 is of interest for biotechnological production of lipid-derived products due to its capability to accumulate high amount of intracellular lipids using various sugars and agro-industrial substrates. Here, we report the genome sequence of the oleaginous A. oryzae BCC7051. The obtained reads were de novo assembled into 25 scaffolds spanning of 38,550,958 bps with predicted 11,456 protein-coding genes. By synteny mapping, a large rearrangement was found in two scaffolds of A. oryzae BCC7051 as compared to the reference RIB40 strain. The genetic relationship between BCC7051 and other strains of A. oryzae in terms of aflatoxin production was investigated, indicating that the A. oryzae BCC7051 was categorized into group 2 nonaflatoxin-producing strain. Moreover, a comparative analysis of the structural genes focusing on the involvement in lipid metabolism among oleaginous yeast and fungi revealed the presence of multiple isoforms of metabolic enzymes responsible for fatty acid synthesis in BCC7051. The alternative routes of acetyl-CoA generation as oleaginous features and malate/citrate/pyruvate shuttle were also identified in this A. oryzae strain. The genome sequence generated in this work is a dedicated resource for expanding genome-wide study of microbial lipids at systems level, and developing the fungal-based platform for production of diversified lipids with commercial relevance.

September 22, 2019  |  

Forward genetics by genome sequencing uncovers the central role of the Aspergillus niger goxB locus in hydrogen peroxide induced glucose oxidase expression.

Aspergillus niger is an industrially important source for gluconic acid and glucose oxidase (GOx), a secreted commercially important flavoprotein which catalyses the oxidation of ß-D-glucose by molecular oxygen to D-glucolactone and hydrogen peroxide. Expression of goxC, the GOx encoding gene and the concomitant two step conversion of glucose to gluconic acid requires oxygen and the presence of significant amounts of glucose in the medium and is optimally induced at pH 5.5. The molecular mechanisms underlying regulation of goxC expression are, however, still enigmatic. Genetic studies aimed at understanding GOx induction have indicated the involvement of at least seven complementation groups, for none of which the molecular basis has been resolved. In this study, a mapping-by-sequencing forward genetics approach was used to uncover the molecular role of the goxB locus in goxC expression. Using the Illumina and PacBio sequencing platforms a hybrid high quality draft genome assembly of laboratory strain N402 was obtained and used as a reference for mapping of genomic reads obtained from the derivative NW103:goxB mutant strain. The goxB locus encodes a thioredoxin reductase. A deletion of the encoding gene in the N402 parent strain led to a high constitutive expression level of the GOx and the lactonase encoding genes required for the two-step conversion of glucose in gluconic acid and of the catR gene encoding catalase R. This high constitutive level of expression was observed to be irrespective of the carbon source and oxidative stress applied. A model clarifying the role of GoxB in the regulation of the expression of goxC involving hydrogen peroxide as second messenger is presented.

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