Abstract
We report here the first genome sequence of the white-rot fungus Obba rivulosa (Polyporales, Basidiomycota), a polypore known for its lignin-decomposing ability. The genome is based on the homokaryon 3A-2 originating in Finland. The genome is typical in size and carbohydrate active enzyme (CAZy) content for wood-decomposing basidiomycetes.
GENOME ANNOUNCEMENT
Obba rivulosa (= Physisporinus rivulosus) is a conifer-dwelling polypore, a selective delignifier that targets lignin over cellulose in the early stages of wood decomposition (1, 2). This makes the species a promising object for studies in lignin degradation and offers prospects for the pretreatment of wood in the pulp industry (3) and bioremediation of soils containing chlorinated compounds (4). Obba rivulosa is a close relative of Gelatoporia subvermispora (= Ceriporiopsis subvermispora), whose draft genome sequence has been published (5). Gelatoporia and Obba belong to the Gelatoporia clade, an evolutionarily distinctive group within Polyporales (Basidiomycota) (6).
North American and Eurasian populations of O. rivulosa differ slightly genetically (7). This genome is based on a monokaryon (3A-2) created from a European strain (T241i, FBCC 949) isolated from a charred conifer log in Finland. Its manganese peroxidases and laccases have been characterized (8–11).
The strain was grown in 2% malt broth. DNA was extracted from manually ground mycelium with the DNeasy plant maxi kit and RNA with RNeasy midi kit (Qiagen). Both the genome and transcriptome were sequenced using Illumina platforms. DNA was sheared to 270 bp using the Covaris E210 (Covaris) and size-selected using SPRI beads (Beckman Coulter). The fragments were treated with end repair, A-tailing, and ligation of Illumina adapters using the TruSeq sample prep kit (Illumina). Stranded cDNA libraries were generated using the TruSeq stranded RNA LT kit. mRNA was purified from total RNA using magnetic beads containing poly(T) oligonucleotides, fragmented, and reverse transcribed using random hexamers and SSII (Invitrogen), followed by second-strand synthesis. The fragmented cDNA was treated with end repair, A-tailing, adapter ligation, and eight cycles of PCR. Both libraries were quantified using Kapa Biosystems’ next-generation sequencing library quantitative PCR (qPCR) kit and run on a Roche LightCycler 480 real-time PCR instrument. The quantified library was then prepared for sequencing on the Illumina HiSeq sequencing platform utilizing a TruSeq paired-end cluster kit, version 4, and Illumina’s cBot instrument to generate clustered flow cells for sequencing on the Illumina HiSeq 2500 using HiSeq and TruSeq SBS sequencing kits, version 4, following a 2 × 150 indexed run recipe.
Genomic reads were filtered for contamination and initially assembled with Velvet (12). The resulting assembly was used to create an in silico long mate-pair library with 3-kbp insert, which was then assembled with the original Illumina library with AllPaths-LG release 46652 (13) to produce the 34.04-Mbp assembly in 712 scaffolds. The transcriptome was assembled using Rnnotator (14). The genome was annotated using the JGI annotation pipeline, resulting in 13,206 predicted genes.
The assembly length and estimated number of genes are typical of saprobic basidiomycetes. Multiple gene models encoding enzymes implicated in lignin degradation are present (nine manganese peroxidases and nine laccases). A single lignin peroxidase gene, but no versatile peroxidase, was detected. Among Polyporales, the carbohydrate active enzyme (CAZy) (15) profile is most similar to that of the closely related Gelatoporia subvermispora.
Accession number(s).
This whole-genome shotgun project has been deposited at DDBJ/ENA/GenBank under the accession no. MAXV00000000. The version described in this paper is MAXV01000000. The annotated genome is available via the JGI fungal genome portal MycoCosm (http://genome.jgi.doe.gov/Obbri1) (16).
ACKNOWLEDGMENT
We thank Petri Oinonen (Ecohelix AB, Stockholm) for creating the monokaryon 3A-2 from the dikaryotic Obba rivulosa strain T241i.
Funding Statement
The work was conducted under the 1000 Fungal Genomes Program by the U.S. Department of Energy Joint Genome Institute, a DOE Office of Science User Facility, supported by the Office of Science of the U.S. Department of Energy under contract no. DE-AC02-05CH11231.
Footnotes
Citation Miettinen O, Riley R, Barry K, Cullen D, de Vries RP, Hainaut M, Hatakka A, Henrissat B, Hildén K, Kuo R, LaButti K, Lipzen A, Mäkelä MR, Sandor L, Spatafora JW, Grigoriev IV, Hibbett DS. 2016. Draft genome sequence of the white-rot fungus Obba rivulosa 3A-2. Genome Announc 4(5):e00976-16. doi:10.1128/.00976-16.
REFERENCES
- 1.Hakala TK, Maijala P, Konn J, Hatakka A. 2004. Evaluation of novel wood-rotting polypores and corticioid fungi for the decay and biopulping of Norway spruce (Picea abies) wood. Enzyme Microb Technol 34:255–263. doi: 10.1016/j.enzmictec.2003.10.014. [DOI] [Google Scholar]
- 2.Maijala P, Kleen M, Westin C, Poppius-Levlin K, Herranen K, Lehto JH, Reponen P, Mäentausta O, Mettälä A, Hatakka A. 2008. Biomechanical pulping of softwood with enzymes and white-rot fungus Physisporinus rivulosus. Enzyme Microb Technol 43:169–177. doi: 10.1016/j.enzmictec.2007.11.017. [DOI] [Google Scholar]
- 3.Hatakka A, Maijala P, Hakala T, Hauhio L, Ellmén J. November 2003. A new white-rot fungus and its use in the pretreatment of wood. Finnish Patent FI 112248.
- 4.Valentín L, Oesch-Kuisma H, Steffen KT, Kähkönen MA, Hatakka A, Tuomela M. 2013. Mycoremediation of wood and soil from an old sawmill area contaminated for since decades. J Hazard Mater 260:668–675. doi: 10.1016/j.jhazmat.2013.06.014. [DOI] [PubMed] [Google Scholar]
- 5.Fernandez-Fueyo E, Ruiz-Dueñas FJ, Ferreira P, Floudas D, Hibbett DS, Canessa P, Larrondo LF, James TY, Seelenfreund D, Lobos S, Polanco R, Tello M, Honda Y, Watanabe T, Watanabe T, Ryu JS, Kubicek CP, Schmoll M, Gaskell J, Hammel KE, St. John FJ, Vanden Wymelenberg A, Sabat G, Splinter BonDurant S, Syed K, Yadav JS, Doddapaneni H, Subramanian V, Lavín JL, Oguiza JA, Perez G, Pisabarro AG, Ramirez L, Santoyo F, Master E, Coutinho PM, Henrissat B, Lombard V, Magnuson JK, Kües U, Hori C, Igarashi K, Samejima M, Held BW, Barry KW, LaButti KM, Lapidus A, Lindquist EA, Lucas SM, Riley R. 2012. Comparative genomics of Ceriporiopsis subvermispora and Phanerochaete chrysosporium provide insight into selective ligninolysis. Proc Natl Acad Sci U S A 109:5458–5463. doi: 10.1073/pnas.1119912109. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Binder M, Justo A, Riley R, Salamov A, Lopez-Giraldez F, Sjökvist E, Copeland A, Foster B, Sun H, Larsson E, Larsson K-H, Townsend J, Grigoriev IV, Hibbett DS. 2013. Phylogenetic and phylogenomic overview of the Polyporales. Mycologia 105:1350–1373. doi: 10.3852/13-003. [DOI] [PubMed] [Google Scholar]
- 7.Miettinen O, Rajchenberg M. 2012. Obba and Sebipora, new polypore genera related to Cinereomyces and Gelatoporia (Polyporales, Basidiomycota). Mycol Prog 11:131–147. doi: 10.1007/s11557-010-0736-8. [DOI] [Google Scholar]
- 8.Hakala TK, Lundell T, Galkin S, Maijala P, Kalkkinen N, Hatakka A. 2005. Manganese peroxidases, laccases and oxalic acid from the selective white-rot fungus Physisporinus rivulosus grown on spruce wood chips. Enzyme Microb Technol 36:461–468. doi: 10.1016/j.enzmictec.2004.10.004. [DOI] [Google Scholar]
- 9.Hakala TK, Hildén K, Maijala P, Olsson C, Hatakka A. 2006. Differential regulation of manganese peroxidases and characterization of two variable MnP encoding genes in the white-rot fungus Physisporinus rivulosus. Appl Microbiol Biotechnol 73:839–849. doi: 10.1007/s00253-006-0541-0. [DOI] [PubMed] [Google Scholar]
- 10.Hildén K, Hakala TK, Maijala P, Lundell TK, Hatakka A. 2007. Novel thermotolerant laccases produced by the white-rot fungus Physisporinus rivulosus. Appl Microbiol Biotechnol 77:301–309. doi: 10.1007/s00253-007-1155-x. [DOI] [PubMed] [Google Scholar]
- 11.Hildén K, Mäkelä MR, Lundell T, Kuuskeri J, Chernykh A, Golovleva L, Archer DB, Hatakka A. 2013. Heterologous expression and structural characterization of two low pH laccases from a biopulping white-rot fungus Physisporinus rivulosus. Appl Microbiol Biotechnol 97:1589–1599. doi: 10.1007/s00253-012-4011-6. [DOI] [PubMed] [Google Scholar]
- 12.Zerbino DR, Birney E. 2008. Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res 18:821–829. doi: 10.1101/gr.074492.107. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Gnerre S, MacCallum I, Przybylski D, Ribeiro FJ, Burton JN, Walker BJ, Sharpe T, Hall G, Shea TP, Sykes S, Berlin AM, Aird D, Costello M, Daza R, Williams L, Nicol R, Gnirke A, Nusbaum C, Lander ES, Jaffe DB. 2011. High-quality draft assemblies of mammalian genomes from massively parallel sequence data. Proc Natl Acad Sci U S A 108:1513–1518. doi: 10.1073/pnas.1017351108. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Martin J, Bruno VM, Fang Z, Meng X, Blow M, Zhang T, Sherlock G, Snyder M, Wang Z. 2010. Rnnotator: an automated de novo transcriptome assembly pipeline from stranded RNA-Seq reads. BMC Genomics 11:663. doi: 10.1186/1471-2164-11-663. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Lombard V, Golaconda Ramulu H, Drula E, Coutinho PM, Henrissat B. 2014. The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res 42:D490–D495. doi: 10.1093/nar/gkt1178. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Grigoriev IV, Nikitin R, Haridas S, Kuo A, Ohm R, Otillar R, Riley R, Salamov A, Zhao X, Korzeniewski F, Smirnova T, Nordberg H, Dubchak I, Shabalov I. 2014. MycoCosm portal: gearing up for 1000 fungal genomes. Nucleic Acids Res 42:D699–D704 doi: 10.1093/nar/gkt1183. [DOI] [PMC free article] [PubMed] [Google Scholar]