Short-Read Sequencing for Genomic Analysis of the Brown Rot Fungus Fibroporia radiculosa

Abstract

The feasibility of short-read sequencing for genomic analysis was demonstrated for Fibroporia radiculosa, a copper-tolerant fungus that causes brown rot decay of wood. The effect of read quality on genomic assembly was assessed by filtering Illumina GAIIx reads from a single run of a paired-end library (75-nucleotide read length and 300-bp fragment size) at three different stringency levels and then assembling each data set with Velvet. A simple approach was devised to determine which filter stringency was "best." Venn diagrams identified the regions containing reads that were used in an assembly but were of a low-enough quality to be removed by a filter. By plotting base quality histograms of reads in this region, we judged whether a filter was too stringent or not stringent enough. Our best assembly had a genome size of 33.6 Mb, an N50 of 65.8 kb for a k-mer of 51, and a maximum contig length of 347 kb. Using GeneMark, 9,262 genes were predicted. TargetP and SignalP analyses showed that among the 1,213 genes with secreted products, 986 had motifs for signal peptides and 227 had motifs for signal anchors. Blast2GO analysis provided functional annotation for 5,407 genes. We identified 29 genes with putative roles in copper tolerance and 73 genes for lignocellulose degradation. A search for homologs of these 102 genes showed that F. radiculosa exhibited more similarity to Postia placenta than Serpula lacrymans. Notable differences were found, however, and their involvements in copper tolerance and wood decay are discussed. R ecent technological achievements in massively parallel sequencing have escalated the rate at which genomes can be sequenced. The most affordable method is the Illumina genome analyzer, which uses reversible dideoxy terminator sequencing chemistry (3). Although the read length is significantly shorter than that with traditional Sanger dideoxy terminator sequencing, Illumina sequencing generates millions of reads, thereby providing the extensive coverage needed to produce an assembly in spite of the higher frequency of base errors and poor read quality. In addition, the read length has increased with technology upgrades, making the technique more attractive for the sequencing of the larger, more complex genomes of eukaryotes. Now that the race to sequence genomes has begun, we are gaining a much clearer picture of how Illumina sequencing performs in practice. Three eukaryotic genomes have been sequenced to date with the Illumina technology alone or in combination with other platforms. These genomes are those of the giant panda bear (29), the plant-pathogenic ascomycete fungus Grosmannia clavigera (10), and another ascomycete, Sordaria macrospora, a model organism for fungal morphogenesis (33). The assembly for the giant panda bear was a formidable feat that most ordinary researchers could not afford to duplicate. It used paired-end reads (52-nucleotide [nt] read length) from 37 Illumina libraries (150-kb to 10-kb insert size) to obtain a 56-fold coverage of the genome (29). The draft assembly e...

Genomic and transcriptomic analyses of the medicinal fungusAntrodia cinnamomeafor its metabolite biosynthesis and sexual development

“…These two brown rot fungi are commonly responsible for the destructive decay of wood in buildings and timbers in the field (46,47). Moreover, both fungi secrete high levels of oxalate and thus show high resistance to copper-based wood preservatives (47). In contrast, A. cinnamommea is an endemic mushroom that is restricted to the endemic aromatic tree Cinnamomum kanehirai Hayata in Taiwan.…”

“…The genomes of two other mushrooms, P. placenta and F. radiculosa, have been sequenced (46,47). These two brown rot fungi are commonly responsible for the destructive decay of wood in buildings and timbers in the field (46,47).…”

Comparative study of genome-wide plant biomass-degrading CAZymes in white rot, brown rot and soft rot fungi

“…We have selected (a) 14 popular white rot fungal strains – Ceriporiopsis subvermispora B (Fernandez-Fueyo et al 2012), Heterobasidion annosum v2.0 (Olson et al 2012), Fomitiporia mediterranea v1.0 (Floudas et al 2012), Phanerochaete carnosa HHB-10118 (Suzuki et al 2012), Pycnoporus cinnabarinus BRFM 137 (Levasseur et al 2014), Phanerochaete chrysosporium R78 v2.2 (Martinez et al 2004; Ohm et al 2014), Dichomitus squalens LYAD-421 SS1 (Floudas et al 2012), Trametes versicolor v1.0 (Floudas et al 2012), Punctularia strigosozonata v1.0 (Floudas et al 2012), Phlebia brevispora HHB-7030 SS6 (Binder et al 2013), Botrytis cinerea v1.0 (Amselem et al 2011), Pleurotus ostreatus PC15 v2.0 (Riley et al 2014; Alfaro et al 2016; Castanera et al 2016), Stereum hirsutum FP-91666 SS1 v1.0 (Floudas et al 2012), Pleurotus eryngii ATCC90797 (Guillen et al 1992; Camarero et al 1999; Ruiz‐Dueñas et al 1999; Matheny et al 2006); (b) 15 popular brown rot fungal strains – Postia placenta MAD 698-R v1.0 (Martinez et al 2009), Fibroporia radiculosa TFFH 294 (Tang et al 2012), Wolfiporia cocos MD-104 SS10 v1.0 (Floudas et al 2012), Dacryopinax primogenitus DJM 731 SSP1 v1.0 (Floudas et al 2012), Daedalea quercina v1.0 (Nagy et al 2015), Laetiporus sulphureus var v1.0 (Nagy et al 2015), Postia placenta MAD-698-R-SB12 v1.0 (Martinez et al 2009), Neolentinus lepideus v1.0 (Nagy et al 2015), Serpula lacrymans S7.9 v2.0 (Eastwood et al 2011), Calocera cornea v1.0 (Eastwood et al 2011), Gloeophyllum trabeum v1.0 (Floudas et al 2012), Fistulina hepatica v1.0 (Floudas et al 2015), Fomitopsis pinicola FP-58527 SS1 (Floudas et al 2015), Hydnomerulius pinastri v2.0 (Kohler et al 2015) and Coniophora puteana v1.0 (Kohler et al 2015); (c) 13 popular soft rot fungal strains – Trichoderma reesei v 2.0 (Martinez et al 2008), Rhizopus oryzae 99-880 from Broad (Ma et al 2009), Aspergillus wentii v1.0 (De Vries et al 2017), Penicillium chrysogenum Wisconsin 54-1255 (Van Den Berg et al 2008), Daldinia eschscholzii EC12 v1.0, Hypoxylon sp. CI-4A v1.0 (Wu et al 2017), Aspergillus niger ATCC 1015 v4.0 (Andersen et al 2011), Hypoxylon sp.…”

Gene Expression Analysis of Copper Tolerance and Wood Decay in the Brown Rot Fungus Fibroporia radiculosa

“…We used high-throughput RNA sequencing (RNA-Seq) as our initial discovery tool, as there are more than 9,000 genes in the genome of F. radiculosa, many of which are members of large gene families with the same functional annotation (48). We then selected 10 of the differentially regulated genes and examined their expression by quantitative reverse transcriptase PCR (qRT-PCR) at 4 time points while the fungus was growing on wood in the presence or absence of MCQ.…”