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Journal of Biotechnology 167 (2013) 142– 155

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Journal of Biotechnology

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Establishment and interpretation of the genome sequence of thephytopathogenic fungus Rhizoctonia solani AG1-IB isolate 7/3/14

Daniel Wibberga,c,1, Lukas Jelonekb,c,1, Oliver Ruppb, Magdalena Henniga, Felix Eikmeyera,c, AlexanderGoesmannb,d, Anton Hartmanne, Rainer Borriss f,g, Rita Groschh, Alfred Pühlera, Andreas Schlütera,∗

a Institute for Genome Research and Systems Biology, CeBiTec, Bielefeld University, D-33501 Bielefeld, Germanyb Computational Genomics, Center for Biotechnology (CeBiTec), Bielefeld University, D-33501 Bielefeld, Germanyc CLIB Graduate Cluster Industrial Biotechnology, CeBiTec, Bielefeld University, D-33501 Bielefeld, Germanyd Bioinformatics Resource Facility, CeBiTec, Bielefeld University, D-33501 Bielefeld, Germanye Research Unit Microbe-Plant Interactions, Helmholtz Zentrum Muenchen, German Research Center for Environmental Health (GmbH), Neuherberg, Germanyf Bakteriengenetik, Institut für Biologie, Humboldt-Universität Berlin, Berlin, Germanyg ABiTEP GmbH, Berlin, Germanyh Leibniz-Institute of Vegetable and Ornamental Crops, D-14979 Großbeeren, Germany

a r t i c l e i n f o

Article history:Received 23 August 2012Received in revised form 3 December 2012Accepted 9 December 2012Available online 29 December 2012

Keywords:Bottom rot pathogenLettuceUltrafast sequencingMitochondrial genomerRNA unit

a b s t r a c t

Anastomosis group AG1-IB isolates of the anamorphic basidiomycetous fungus Rhizoctonia solani Kühnaffect various agricultural and horticultural important crops including bean, rice, soybean, figs, hortensia,cabbage and lettuce. To gain insights into the genome structure and content, the first draft genomesequence of R. solani AG1-IB isolate 7/3/14 was established. Four complete runs on the Genome Sequencer(GS) FLX platform (Roche Applied Science) yielding approx. a 25-fold coverage of the R. solani genomewere accomplished. Assembly of the sequence reads by means of the gsAssembler software version2.6 applying the heterozygotic mode resulted in numerous contigs and scaffolds and a predicted sizeof 87.1 Mb for the diploid status of the genome. ‘Contig-length vs. read-count’ analysis revealed thatthe assembled contigs can be classified into five different groups. Detailed BLAST-analysis revealed thatmost contigs of group II feature high-scoring matches to other contigs of the same group suggesting thatdistinguishable allelic variants exist for many genes. Due to the supposed diploid and heterokaryoticnature of R. solani AG1-IB 7/3/14, this result has been anticipated. However, the heterokaryotic characterof the isolate is not really supported by sequencing data obtained for the isolate R. solani AG1-IB 7/3/14.Coverage of group III contigs is twice as high as for group II contigs which can also be explained by thediploid status of the genome and indistinguishable alleles on homologous chromosomes. Assembly ofsequence data led to the identification of the rRNA unit (group V contigs) and the mitochondrial (mt)genome (group IV contigs) which is a circular molecule of 162,751 bp in size featuring a GC-content of36.4%. The R. solani 7/3/14 mt-genome is one of the largest fungal mitochondrial genomes known todate. Its large size essentially is due to the presence of numerous non-conserved hypothetical ORFs andintrons. Gene prediction for the R. solani AG1-IB 7/3/14 genome was conducted by the Augustus GenePrediction Software for Eukaryotes (version 2.6.) applying the parameter set for the fungus Coprinopsiscinerea okayama 7#130. Gene prediction and annotation provided first insights into the R. solani AG1-IB 7/3/14 gene structure and content. In total, 12,422 genes were predicted. The average number ofexons per gene is five. Exons have a mean length of 214 bp, whereas introns on average are 66 bp inlength. Annotation of the genome revealed that 4169 of 12,422 genes could be assigned to KOG functionalcategories.

© 2012 Elsevier B.V. All rights reserved.

∗ Corresponding author. Tel.: +49 521/106 8757; fax: +49 521/106 89041.E-mail address: [email protected] (A. Schlüter).

1 Both authors contributed equally to this work.

1. Introduction

The fungus Rhizoctonia solani Kühn (teleomorph Thanatepho-rus cucumeris [Frank] Donk) is responsible for many economicallyimportant plant diseases worldwide (Sneh et al., 1996). Donk(1956) initially proposed to designate the genus Thanatephorus asthe teleomorphic phases of the R. solani multinucleate anamorph.

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D. Wibberg et al. / Journal of Biotechnology 167 (2013) 142– 155 143

It is generally accepted that the genus applies to most parasiticfungi like R. solani considered as a very destructive plant pathogen(González-Garcia et al., 2006). R. solani is a species complex ofgenetically distinct fungi groups in the cantherelloid clade of thephylum Basidiomycota (Binder et al., 2005). Isolates of this speciescomplex were classified into genetically distinct groups namedanastomosis groups (AGs) based on hyphal interaction betweenisolates of the same AG. In general, classification systems of Rhizoc-tonia species are based on anamorphic features. Occurrence of thephenomenon ‘anastomosis’ is the primary mechanism of geneticexchange in Rhizoctonia. Hence, isolates within an AG are closelyrelated to one another and tend to have a similar host range. Cur-rently, 13 AGs (AG1 to AG13) featuring distinctive physiology andgenetic composition have been recognized (Carling et al., 2002).Moreover, some AGs such as AG1 and AG2 are further divided intogenetically distinct sub-groups (Ogoshi, 1987). It has been estab-lished that all AGs and their sub-groups are genetically isolatedand represent highly divergent evolutionary units (Rosewich et al.,1999; Ceresini et al., 2007). R. solani isolates of certain AGs such asAG3, AG5 and AG8 feature a limited host range whereas membersof other AGs including isolates of AG1 and AG2 and its sub-groupsare able to infect various host plants. AG1 isolates are subdividedinto six sub-groups, AG1-IA, -IB, -IC, -ID, -IE and -IF based on differ-ences in colony morphology, biochemical, genetic and pathogeniccharacteristics (Priyatmojo et al., 2001; Ogoshi, 1987). R. solaniAG1 is a major pathogen on economically important Poaceae sta-ple crops worldwide including rice and maize (Pascual et al., 2000;Jones and Belmar, 1989), on Rubiaceae including coffee (Priyatmojoet al., 2001), and on Papilionaceae including soybean (Yang et al.,1990). Crops like maize, rice and soybean are of high importancefor human nutrition. In addition to diseases on bean, rice, soybean,hortensia and leguminous woody plants (González-Garcia et al.,2006), isolates of R. solani AG1-IB are known to infect crops ofthe family Brassicaceae (cabbage) (Mukou et al., 1975), Asteraceae(lettuce) (Herr, 1992; Grosch et al., 2004) and Basellaceae (Indianspinach) (Baiswar et al., 2012). R. solani has been found on all typesof lettuce and occurs wherever lettuce is cultivated (Davis et al.,1997). AG1-IB isolates from lettuce are able to infect various veg-etable crops including bean, broccoli, cabbage, carrot, radish andspinach (Grosch et al., 2004). In summary, isolates of the sub-groupAG1-IB are able to parasitize on a wide range of hosts worldwideand dissemination for instance occurs by means of seeds (Godoy-Lutz et al., 1996).

As a fundamentally asexual fungus the anamorph R. solani doesnot reproduce sexually (Adams, 1996). The pathogen survives anddisseminates in form of sclerotia and/or vegetative mycelia (Keijeret al., 1996). However, Rhizoctonia species are strong saprophytesand able to survive in the absence of host plants by feeding onorganic matter (Sneh et al., 1996). In general, the infection pro-cess of R. solani includes adhesion, penetration, colonization, andhost reaction. Isolates of the fungus infect a host plant mostly byformation of infection cushions such as described for AG1 isolateson rice (Matsuura, 1986). However, also the production of lobateappressoria was observed on rice (Marshall and Rush, 1980). Theinfection process is promoted by the production of pectinolytic andcellulolytic enzymes. The production of endopectinlyase has beenreported to be associated with tissue degradation in later stages ofinfection (González-Garcia et al., 2006). Initial infection on lettucestarts with small rust colored to brown spots on lower midribs. R.solani moves up the plant by surface hyphae and forms new infec-tion structures. Under favorable conditions for the disease, R. solaniinfection can spread rapidly into the lettuce head, causing rot ofmidribs and leaves. Finally, the infection process can result in severenecrotic damage of lettuce heads. In a later stage, yellow-brownmycelium and brown sclerotia appear on infected tissue (Blancardet al., 2006).

The fungus R. solani reigns among the most serious threatsto food production for many economically important crops. Theavailability of effective control strategies to reduce the potentialimpacts of diseases caused by R. solani currently is limited. There-fore, the understanding of the molecular mechanisms of R. solaniinfection and pathogenesis will allow the identification of essen-tial pathogenicity genes and hence will enable the developmentof suitable management strategies. However, much of the genet-ics of the anamorphic fungus R. solani remains unknown until now.Hyphae are multinucleate and primarily heterokaryotic (Yang et al.,1992; Julian et al., 1996; Toda and Hyakumachi, 2006). In general,it is assumed that field isolates are heterokaryotic and that theunlike nuclei of heterokaryotic isolates fuse to form diploid nuclei(Rosewich et al., 1999).

The number of nuclei in AG1-IB isolates varies from 3 to 21 withan average of six per cell (Grosch et al., 2004). Because of the mult-inucleate and heterokaryotic nature, genetic studies addressinghost-pathogen interaction are relatively scarce. Hence, sequenc-ing of the R. solani AG1-IB genome was initiated to improve theknowledge on genetic determinants having a function in diseasemanifestation.

Recently, the draft genome sequence of a R. solani AG3 isolatefrom potato became available (Cubeta et al., 2009). Applying high-throughput sequencing, a 50 Mb draft genome was establishedfor this isolate (http://www.rsolani.org). Based on this knowledge,the first genome project was initiated for a R. solani AG 1-IBisolate.

The AG1-IB isolate 7/3/14 chosen for sequence analysis ori-ginates from a lettuce plant with bottom rot symptoms andwas shown to be highly aggressive on lettuce and other veg-etable crops such as bean, cabbage and carrot (Grosch et al.,2004). In contrast to AG3 isolates, AG1-IB representatives are ableto infect a broad range of plant species as described above. Inthis study, the genome sequence of R. solani AG1-IB 7/3/14 isreported. Gene prediction and annotation were established forthe R. solani AG1-IB 7/3/14 genome. Predicted genes were ana-lyzed with emphasis on their exon–intron structure. The presentstudy was started to learn about the size, structure, function andgene content of the R. solani AG1-IB isolate 7/3/14 genome. Inaddition, questions concerning the isolate’s nuclear condition andthe degree of its heterokaryotic character should be addressed.Finally, the assembly of the R. solani AG1-IB 7/3/14 mitochondrialgenome from whole-genome-shotgun sequence data should beapproached.

2. Material and methods

2.1. Origin and growth of R. solani AG1-IB 7/3/14

The strain R. solani AG1-IB 7/3/14 (AJ868459) was isolated from a lettuce plant(Lactuca sativa) with bottom rot symptoms in a field at the IGZ (Großbeeren,Germany, 52◦33′N, 13◦22′O) and characterized by conventional and molecular tools(Grosch et al., 2004, 2007). This strain causes severe symptoms on lettuce. Thestrain was stored at −25◦C and routinely grown on potato-dextrose agar (PDA; VWRInternational GmbH, Berlin Germany).

2.2. 454-Pyrosequencing of the R. solani AG1-IB 7/3/14 genome and genomeassembly

Genomic DNA of R. solani AG1-IB 7/3/14 for 454-pyrosequencing was isolated asdescribed previously (Grosch et al., 2007). To confirm the identity of the DNA, a 18SrDNA-specific region was amplified and sequenced. BLASTn analysis revealed thatthe isolate’s 18S rDNA-sequence is highly related to corresponding sequences origi-nating from species of the genus Rhizoctonia. The quality of the DNA was assessed bygel-electrophoresis and the quantity was estimated by a fluorescence-based methodusing the Quant-iT PicoGreen dsDNA kit (Invitrogen) and the Tecan Infinite 200Microplate Reader (Tecan Deutschland GmbH).

Two whole-genome-shotgun libraries were generated using 5 �g of genomicDNA and the GS FLX Titanium General Library Preparation Kit (Roche) accordingto the protocol provided by the manufacturer. Secondly, a 3K Long-Tag Paired Endlibrary was generated using 10 �g of genomic DNA and the GS FLX Paired-end Library


144 D. Wibberg et al. / Journal of Biotechnology 167 (2013) 142– 155

Preparation Kit (Roche) according to the manufacturer’s protocol. Paired-end readswere used to determine the orientation and relative positions of contigs generatedby the de novo shotgun sequencing and assembly. The DNA libraries were amplifiedby emulsion PCR and sequenced applying the Titanium sequencing chemistry onfour PicoTiterPlates (454 Life Sciences, Roche). Upon sequencing and processing ofthe raw data, a de novo assembly was performed using the GS De Novo Assem-bler software release version 2.6 with heterozygotic mode and default settings.The heterozygotic mode was used to take into account that the sequence readsoriginate from a diploid organism. The assembled draft sequence for the R. solaniAG1-IB genome was deposited in the GenBank database under the accession nos.CAOJ01000001–CAOJ01023355 and HF546977.

2.3. Statistical contig information

First insights into the quality of the assembly were provided by calculatingthe read coverage for each contig. For each contig from the set of n con-tigs C = {ci|i ∈ {1..n}} the raw coverage is calculated by the equation covraw(ci) =(readcount(ci))/readcount(ci). The raw coverage is then normalized to the medianraw coverage of all contigs longer than 10,000 bp, as these contigs are expected tohave a single fold coverage, i.e. C = {covraw(cj)|∀j : length(cj) ≥ 10, 000} and cov(ci) =readcount(ci)/(length(ci) × median(C)). Coverage values between 0.5 and 1.5 indi-cate that the corresponding contigs are represented once in the genome; ratios lowerthan 0.5 indicate underrepresented contigs and ratios higher 1.5 indicate overrepre-sented contigs. ‘Contig-length vs. read-count’ plot analyses were described in moredetail previously (Heinl et al., 2012; Schwientek et al., 2012).

2.4. Analysis of local similarities between R. solani AG1-IB 7/3/14 contigs

Genomic contigs resulting after assembly were analyzed for large local similar-ities applying the BLASTn algorithm (Altschul et al., 1990). For each group of contigseach contig longer than 1000 bp was compared to a database including all othercontigs from the same group. Identical hits, hits with an e-value above 1 × 10−10,hits possessing a sequence identity less than 90% and hits featuring a match lengthlower than 1000 bp were removed. The obtained data were used to estimate thenumber of allelic variants within the R. solani AG1-IB 7/3/14 genome.

2.5. Identification, finishing and analysis of the R. solani AG1-IB 7/3/14mitochondrial genome

For identification and assembly of the R. solani AG1-IB 7/3/14 mitochondrialgenome different methods were used. First, the GC content of all scaffolds wasanalyzed to identify scaffolds deviating from the average GC-content. In gen-eral, mitochondria of Basidiomycetes feature a lower GC content compared to thechromosomes (Villa and Storck, 1968). Secondly, mitochondrial scaffolds shouldbe over-represented in the genome, since most eukaryotic cells contain multiplemitochondria (Ballard and Whitlock, 2004). Data analysis revealed that only twoscaffolds represent the R. solani AG1-IB 7/3/14 mitochondrial genome. After qualitycheck, all reads of the identified mitochondrial contigs were extracted and reassem-bled using the GS De Novo Assembler software (release version 2.6.) applying defaultsettings. Sequence gaps were closed in silico based on contig graph informationcreated by the GS De Novo Assembler. Subsequently, automatic gene predictionand annotation were performed using the genome annotation system GenDB 2.0(Meyer et al., 2003). The gene identification strategy Reganor (McHardy et al., 2004)was used which combines the results of Glimmer 2 (Delcher et al., 1999) and Crit-ica (Badger and Olsen, 1999). Intergenic regions were analyzed for missed codingsequences by BLAST programs (Altschul et al., 1997). Putative ribosomal bindingsites, tRNA genes and rRNA genes were identified with RBSFINDER (Suzek et al.,2001), tRNAscan-SE (Lowe and Eddy, 1997) and RNAweasel (Lang et al., 2007). Priorto the manual annotation, an automatic annotation was computed based on theresults of different tool: tools similarity searches were performed against differentdatabases including SWISS-PROT and TrEMBL, KEGG, Pfam, TIGRFAM and InterPro.Additionally, SignalP, helix-turn-helix and TMHMM were applied. Finally, each genewas functionally classified by assignment of a Cluster of Orthologous Groups (COG)number and its corresponding COG category (Tatusov et al., 2003) and Gene Ontol-ogy terms (Harris et al., 2004). After automatic annotation, sequence informationwas refined manually. DNA and deduced amino acid sequences of the mt-genomewere compared to related mitochondrial genomes stored in the NCBI database byapplying BLASTn, BLASTp and BLASTx (Altschul et al., 1990). The mitochondrialgenome plot was drawn as described before (Szczepanowski et al., 2011; Eikmeyeret al., 2012).

2.6. Phylogenetic analyses

Phylogenetic classification of R. solani AG1-IB 7/3/14 was done as follows: (i)the phylogenetic position of R. solani AG1-IB 7/3/14 was assessed according to anapproach outlined previously (Swann and Taylor, 1993) using the 18S rRNA geneas a marker. Nucleotide sequences of 18S rRNA genes were extracted from corre-sponding database entries (Table 1A) and analyzed by using the Neighbor-Joiningalgorithm (Saitou and Nei, 1987; Studier and Keppler, 1988). (ii) Phylogeneticclassification of R. solani strains was further refined by analyzing the 18S rRNA gene

Table 1Sequences included in the 18S rRNA gene phylogenetic analysis.

(A) Strains Accession number

Lentinula edobes FJ379277M. perniciosa AY916739S. commune X54865C. cinerea M92991L. bicolor JQ753771S. lacrymans AJ440946Phaerochaete chrysosporium AF026593R. solani AG1-IB 7/3/14 AJ868459Malassezia globosa EU192364Tremella mesenterica JN043521Crytococcus bacillispora AY083224Crytococcus neoformans AJ560321Rhodotorula graminis HM371376

(B) Strains Accession number

R. solani AG1-IB 7/3/14 AJ868459R. solani AG1-IA D85629R. solani AG1-IB D85630R. solani AG7 D85641R. solani AG1-IC D85631R. solani AG4 HG-I D85636R. solani AG6 Gv D85640R. solani AG6 HG-I D85639R. solani AG4 HG-II D85637R. solani AG5 D85638R. solani AG2-2 IV D85634R. solani AG2-2 IIIB D85633R. solani AG-BI D85645R. solani AG3 D85635R. solani AG8 D85642R. solani AG2-1 D85632R. solani AG9 TP D85643R. solani AG9 TX D85644Rhizoctonia sp. CPCC 480725 FJ515314Rhizoctonia sp. AG-A D85646

by the Neighbor-Joining algorithm using previously described settings (Kuninaga,unpublished) (Table 1B). A bootstrap analysis using 1000 re-samplings of thesequence data was carried out.

2.7. Comparison of the R. solani AG1-IB 7/3/14 draft genome to the genomes ofrelated fungi

Sequence based comparisons between the R. solani AG1-IB 7/3/14 genome andthe genomes of other fungi were performed using the computer program r2cat rep-resenting a software framework for reference genome mapping (Husemann andStoye, 2010). This program visualizes possible rearrangements and core regions inthe R. solani AG1-IB 7/3/14 genome in comparison to reference sequence data ofCoprinopsis cinerea okayama7#130 (Stajich et al., 2010), Laccaria bicolor S238N-H82(Martin et al., 2008), Schizophyllum commune H4-8 (Ohm et al., 2010), and Serpulalarymans S7.9 (Eastwood et al., 2011).

2.8. Gene prediction and genome annotation

Gene prediction was conducted by applying Augustus version 2.6 (Stanke et al.,2006) with the parameter sets for C. cinerea and L. bicolor. A comparison of the pre-dictions with cuffdiff from the cufflinks package (Trapnell et al., 2010) revealed thatboth predictions comprise a large amount of loci that overlap. During comparativeand taxonomic analysis, C. cinerea was found to be the most appropriate referencesequence. Accordingly, the C. cinerea prediction was used for further analysis. Geneswere functionally annotated using a modified version of the genome annotationsystem GenDB 2.0 (Meyer et al., 2003) designed to handle eukaryotic genomes.Automatic annotation was computed based on the tool BLASTp (Altschul et al.,1990). Similarity searches were performed against different databases includingKEGG, KOG and SWISS-PROT (Tatusov et al., 2003; Kanehisa et al., 2004; Boeckmannet al., 2003). Putative tRNA genes were identified with tRNAscan-SE (Lowe and Eddy,1997).

The identified genes have been assigned to taxonomic groups based on a BLASTsearch against the nr database. The taxonomic information was retrieved for thebest BLAST hit with an e-value cutoff of 1 × 10−5 and a minimum sequence identityof 40%.



2.9. Validation of gene predictions

The predicted R. solani AG1-IB 7/3/14 gene products were blasted applyingBLASTp (Altschul et al., 1990) against the SWISS-PROT database (Boeckmann et al.,2003) with an e-value cutoff of 1 × 10−4 to identify highly conserved proteins. Tar-gets with the highest query coverage representing glycolysis or the citrate cycleenzymes (based on EC numbers from the appropriate KEGG pathways (Kanehisaet al., 2004)) were compiled in a new query database. This database was mappedwith blat (Kent, 2002) against gene predictions applying the L. bicolor or the C. cinereagene model. The best hit in terms of non-overlapping consecutive predictions andthe highest number of matches was compared between both predictions. Both genepredictions showed identical or almost identical results.

3. Results and discussion

3.1. R. solani isolate 7/3/14 belongs to the anastomosis groupAG1-IB based on 18S-rRNA gene sequencing

To gain insights into the molecular basis underlying infectionand pathogenesis processes, complete genome sequencing of thefungus R. solani AG1-IB isolate 7/3/14 was accomplished. It isexpected that analysis and interpretation of genome sequence datawill promote the development of improved and effective diseasecontrol strategies. The R. solani AG1-IB isolate 7/3/14 was chosenfor complete nucleotide sequencing because of its high aggressive-ness and pathogenicity on different host plants. It infects amongother plants lettuce, spinach, bean, radish, broccoli and kohlrabi(Grosch et al., 2004).

To confirm assignment of the R. solani isolate 7/3/14 to the AG1-IB group (Grosch et al., 2007) and to infer its relationship to otherfungi of the phylum Basidiomycota on a molecular basis, its partial18S rRNA gene sequence was determined. Classification of the can-didate isolate by means of the Neighbor-Joining algorithm (Saitouand Nei, 1987; Studier and Keppler, 1988) comprising selected ref-erence sequences demonstrated that the resulting phylogenetictree is divided into seven different groups at the order level (Fig. 1A).The candidate isolate clusters together with sequences originatingfrom a R. solani AG1-IB reference isolate (accession No. D85630)and Phanerochaete chrysosporium (accession no. AF026593). Thelatter fungus is known as wood- and plant-destroying organism.Completely sequenced closest relatives of R. solani AG1-IB 7/3/14are the ectomycorhizal fungus L. bicolor and the basidiomyce-tous fungi C. cinerea, S. commune and Serpula lacrymans (Fig. 1A).Accordingly, these species are tentative candidates for compar-ative genome analysis and reference genome mapping. A moredetailed phylogenetic classification of the isolate 7/3/14 on thespecies level revealed that it clusters together with a R. solani AG1-IB reference sequence (accession no. D85630) as can be seen inFig. 1B.

3.2. The draft genome sequence of R. solani AG1-IB isolate 7/3/14.

To determine the complete genome sequence of the R. solani iso-late 7/3/14, high throughput sequencing on the Genome Sequencer(GS) FLX system was applied. The genome size of R. solani AG3was estimated to vary from 52.4 to 71.5 Mb depending on themethod of analysis (Cubeta et al., 2009). Accordingly, four com-plete sequencing runs on the GS FLX platform were carried out tosufficiently cover the R. solani AG1-IB 7/3/14 genome. One of theseruns was applied to sequence a 3K paired-end library to enablescaffolding of contigs in sequence assemblies. Four sequencingruns resulted in 6,970,249 reads yielding approx. 2.2 Gb sequenceinformation. A de novo assembly of the obtained sequence reads bymeans of the Newbler Assembly software (version 2.6.) applyingthe heterozygotic mode generated 17,517 large contigs (≥500 bp)and 5905 small contigs (<500 bp) accounting for a total length of48.62 Mb (Table 2). The relatively high number of contigs could

not significantly be reduced even after the fourth sequencingrun (see Table 2) suggesting that intrinsic characteristics of thegenome prevent further joining of contigs in assemblies. Hetero-geneity in the genome, repetitive elements and sequences suchas microsatellites could be responsible for this observation. The R.solani AG1-IB 7/3/14 draft genome features a GC-content of 48.1%which is slightly higher than the GC-content of 46.1–47.6% thatwas determined for AG1-IB based on DNA thermal melting profiles(Kuninaga and Yokosawa, 1985a,b). The largest contig has a sizeof approximately 80 kb. Assembled contigs could be arranged into1600 scaffolds consisting of altogether 18,085 scaffolded contigs.The largest scaffold has a size of about 1 Mb. About 96% of all readswere included in the assembly and the estimated genome sizewas calculated to be approximately 87.2 Mb which nearly is twiceas high as the size estimated for this AG by Keijer et al. (1996).The large predicted genome size of the isolate sequenced in thisstudy might be due to its diploid nature, whereas the genome sizeestimated by Keijer et al. (1996) refers to the haploid genome. Thegenome size of R. solani AG1-IB isolate 7/3/14 is in accordance withresults obtained within a genome project for an isolate belongingto the R. solani AG3 (http://www.rsolani.org).

3.3. Differentiation of R. solani AG1-IB isolate 7/3/14 contigsbased on contig-length vs. read-count analysis

To gain deeper insights into the R. solani AG1-IB 7/3/14 genomecomposition, a ‘contig-length vs. read-count’ plot was calculated(Fig. 2A). This plot format represents all contigs as a function oftheir length and the corresponding amount of assembled reads.Obtained results are presented by a point cloud (Fig. 2A). How-ever, appearance of different contig groups was apparent. Usually,three different groups of contigs can be observed in these kindof plots: unique, overrepresented and underrepresented contigs.However, in the case of R. solani AG1-IB, assembled contigs canbe classified in five different groups: (I) underrepresented contigshaving a coverage of below 0.5×, (II) contigs featuring a coverageof 0.5× to 1.5×, (III) contigs showing a coverage between 1.5× and3×, (IV) contigs with a coverage from 3× to 50× and (V) contigswith a coverage above 50× (Table 3). Contigs assigned to thesefive groups were analyzed by applying the BLAST algorithm todeduce further information on this specific plot structure. Onlyeight of 211 underrepresented contigs of group I produced hitsagainst the NCBI database. All of these hits refer to different fun-gal genomes and thus may represent regions that posed difficultiesin the sequencing approach. However, assembly artefacts, espe-cially for those contigs that are not related to any fungal referencesequences, cannot be excluded. Contigs of the two groups II andIII represent chromosomal sequences, because they mostly encodetypical housekeeping genes. Contigs of these two groups almostrepresent the complete chromosomal R. solani 7/3/14 genome (ca.97% of the total haploid genome size, see Table 3). Group II com-prises contigs, the sequences of which are sufficiently differentand therefore were assembled into individual contigs (Fig. 2B).Group III consists of contigs representing identical or almost iden-tical allelic variants in the sequenced diploid organism (Fig. 2B).The assembler was not able to split sequences of almost identi-cal allelic variants into two contigs. Accordingly, contigs of thisgroup feature a coverage of approximately two-fold. The contigsof groups IV and V were mostly allocated to the more abundantmitochondrial DNA (group IV) or the most abundant DNA encodingribosomal RNAs (rRNA), (group V). Interestingly, contigs belongingto two large scaffolds and featuring coverage values of 15× to 50×feature significantly lower GC-content values. These two scaffoldscould be assigned to the R. solani 7/3/14 mitochondrial genome (seebelow).



A

B

Fig. 1. Taxonomic classification of the isolate R. solani AG1-IB 7/3/14. (A) Phylogenetic classification of R. solani AG1-IB 7/3/14 in relation to members of the phylumBasidiomycota. The phylogenetic tree is based on 18S rRNA gene sequences applying default settings of the Neighbor-Joining algorithm. Different taxonomic groups at orderlevel are indicated. Completely sequenced members of the phylum Basidiomycota were marked in purple. (B) Phylogenetic analysis of isolates belonging to the speciesR. solani according to Kuninaga (unpublished) based on 18S rRNA gene sequences is presented. The phylogenetic tree was calculated by applying default settings of theNeighbor-Joining algorithm. The bar below the phylogenetic trees represents the scale of sequence divergence. Numbers at branches represent the percentage of congruentclusters in 1000 bootstrap trials when the values were greater than 50%.



Table 2Sequencing and assembly statistics of the R. solani AG1-IB isolate 7/3/14 genome.

Run 1a Run 1 + 2a Run 1 + 2 + 3a Run 1 + 2 + 3 + 4a

Reads 1,494,801 3,150,478 4,838,458 6,970,249Bases 602,362,963 1,235,713,288 1,800,472,310 2,180,219,455Aligned Reads 1,468,124 (98.22%) 3,131,173 (98.38%) 4,760,743 (98.39%) 6,693,785 (96.03%)Contigs 23,815 25,995 23,137 23,422Large Contigs 15,393 18,858 17,675 17,517Largest Contig 66,929 bp 79,263 bp 79,195 bp 79,195 bpContig size 43.9 Mb 46.7 Mb 47.9 Mb 48.6 MbScaffolds – – – 1600Largest Scaffold – – – 1,082,565 bpScaffold size – – – 47.65 MbEstimated genome size 60.2 Mb 83.4 Mb 90.0 Mb 87.2 Mb

a Run 1, 2, 3: whole-genome shotgun sequencing on the Genome sequence (GS) FLX platform; Run 4: paired end sequencing on the GS FLX platform.

Fig. 2. Contig-length vs. Read-count plot of R. solani AG1-IB 7/3/14 contigs. (A) The length of contigs (x-axis) was plotted as a function of the number of reads assembled intoeach contig (y-axis). Lines indicate the predicted coverage of contigs within the genome. The red line represents the calculated coverage of unique contigs (one-fold). Thedifferent colors of contigs represent five different groups: Underrepresented contigs having a coverage of below 0.5× (black), contigs featuring a coverage of 0.5× to 1.5×(blue), contigs showing a coverage between 1.5× and 3× (orange), contigs with a coverage from 3× to 50× (green) and contigs located in the top left area of the plot (red).(B) Differentiation of group II and III contigs as recognized in the Contig-length vs. Read-count plot. First, a homologous chromosome pair (ChrZ-A and ChrZ-B) featuring twoallelic marker genes each is presented schematically. Group II comprises contigs (different shades of yellow, A and C), the sequences of which are sufficiently different inlength and sequence identity. Group III (grey rectangles, B) consists of contigs representing identical or almost identical allelic variants in the sequenced diploid organism.

Table 3Statistics of the contig-length vs. read count analysis.

Group Ia Group IIa Group IIIa Group IVa Group Va

Ratiob <0.5× 0.5× to 1.5× 1.5× to 3× 3× to 50× >50×Total number of contigs 211 11,505 8475 3196 35Largest contig 1281 bp 53,482 bp 79,195 bp 34,997 bp 4215 bpPercentage of all contigs 0.90% 49.12% 36.18% 13,64% 0.14%Total number of reads 64,794 2,665,531 2,963,196 747,540 139,346Total contig length 62,927 bp 30,667,998 bp 16,448,439 bp 1,425,512 bp 12,483 bpAverage contig length 298 bp 2666 bp 1941 bp 446 bp 357 bp

a Contigs were subdivided into groups based on the contig-length vs. read-count plot analysis (Fig. 2).b Ratios were calculated using the equations described in chapter 2.3.



Table 4Local similarities within the five groups (I to V) of contigs.a

Maximal match (bp) Group I Group II Group III Group IV Group V

1001–5000 0 2999 158 6 05001–10,000 0 402 0 0 0>10,000 0 24 0 0 0

Total hits 0 (4)b 3425 (7695)b 158 (4402)b 6 (123)b 0 (3)b

a Contigs were grouped according to the contig-length vs. read-count analysis (see Fig. 2 and Table 2). Contigs with a length >1000 bp were searched for local similaritiesto other contigs within their group. The table shows the number of regions that feature a high similarity to contigs of the same group.

b In brackets the total number of sequences larger than 1000 bp for each group is shown.

3.4. The diploid character of R. solani AG1-IB isolate 7/3/14accounts for contigs featuring local similarities

The R. solani AG1-IB 7/3/14 genome was sequenced in its nat-ural state. Hence, alleles of genes are expected to be present inthe sequence dataset. To analyze whether the assembled R. solani7/3/14 contigs feature local similarities to other contigs within thedataset, a BLAST search of all contigs against all contigs of thesame group (I–V, see Fig. 2) was performed. It was observed thata large number of contigs within group II shares similar regionswith lengths of more than 1000 bp with at least one other contigof the same group (see Table 4). In contrast, local similarities weredetected less frequently within the other groups.

The largest matching region of a group II contig is longer than20 kb featuring a sequence identity of 94% to another contig of thesame group. It is very likely that this contig pair contains allelicvariants of genes that are significantly different to each other andtherefore were not assembled into one contig (see Fig. 3). Occur-rence of sequence differences is not evenly distributed over thealigned and matching contig segments indicating that some regionsare more conserved than others (see Fig. 3).

Local similarities between contigs of one group in first instanceconfirm the diploid character of the genome. Previously, gene flowand the degree of heterozygosity were analyzed for a R. solani AG1-IA isolate based on single-copy nuclear RFLP markers. An excess ofheterozygotes of the diploid organism was observed for certain loci(Rosewich et al., 1999). Likewise, in this study it was shown that dif-ferent alleles of genes exist in the R. solani 7/3/14 genome. However,it cannot be excluded that allelic variants of genes are located onhomologous chromosomes in different nuclei since the sequencedR. solani isolate is supposed to be heterokaryotic. Presence of mul-tiple copies for 141 group II contigs featuring shared regions largerthan 1000 bp and more than 90% sequence identity also may beexplained by an heterokaryotic character of the R. solani AG1-IB7/3/14 genome. However, the heterokaryotic character of the iso-late 7/3/14, is not strongly supported by the sequencing data or inother words there only seems to be limited heterogeneity betweendifferent nuclei.

3.5. Assembly and annotation of the R. solani AG1-IB isolate7/3/14 mitochondrial genome

Scaffolds representing the mitochondrial (mt) genome wereidentified within the R. solani AG1-IB 7/3/14 genome dataset. Readsof the mitochondrial contigs were extracted and reassembled bymeans of the Newbler Assembly software (version 2.6.) usingdefault setting. The separately assembled mitochondrial readsresulted in the formation of 31 contigs. To finish the draft sequenceof the R. solani AG1-IB 7/3/14 mitochondrial genome, an in silicobased strategy was applied. By means of Newbler contig graphinformation, 25 of 31 gaps could be closed while the sequences ofsix gaps remained undefined due to highly repetitive A + T spacersequences. The resulting improved draft sequence of the mito-chondrial genome was automatically annotated using GenDB 2.0

(Meyer et al., 2003). Subsequently, sequence interpretation wasrefined manually by means of BLAST and splice-site analyses. Thefinal mitochondrial consensus sequence is 162,751 bp in size fea-turing a coverage depth of about 70-fold. By means of BLAST andthe GenDB gene prediction pipeline, 28 protein coding sequencescould be identified as well as 25 sequences encoding tRNAs for all 20amino acids. Moreover, small and large rRNA loci are present in thegenome (Fig. 4). Accordingly, the R. solani AG1-IB 7/3/14 mitochon-drial genome is one of the largest fungal mitochondrial genomesknown to date. Interestingly, the main reason for the large size ofthe R. solani AG1-IB 7/3/14 mitochondrial genome is the presence ofnon-conserved hypothetical ORFs and numerous introns. A similarexplanation was given for the large mitochondrial genome size ofthe ascomycete saprophytic fungus Podospora anserina (Cummingset al., 1990) and the basidiomycete hemibiotrophic fungus Monil-iophthora perniciosa (Formighieri et al., 2008). Based on sequencecomparisons of the mitochondrial small subunit ribosomal RNAgene (16S rRNA gene, rns) with bacterial 16S rRNA genes, the R.solani AG1-IB 7/3/14 mitochondrial genome might originate froma bacterium related to ancestors of the genus Borrelia. The mito-chondrial 16S rRNA shows the highest degree of similarity (72%) toBorrelia tanukii strain Hk501. In comparison to the chromosomalDNA, the GC-content of the mitochondrial genome is relatively low(36.4% vs. 48.1%). Commonly, GC-skew analysis is applied to iden-tify replication origins in bacterial genomes (Grigoriev, 1998). Asimilar approach has been proposed for the prediction of the originin fungal mitochondrial genomes (Formighieri et al., 2008). How-ever, for the R. solani AG1-IB 7/3/14 mitochondrial genome it wasnot possible to predict the origin of replication based on GC-contentor GC-skew analyses. In summary, compilation of the mitochon-drial genome from whole genome shotgun sequence data was verymuch facilitated by exploiting results obtained in contig-length vs.read-count plot analyses.

3.6. Gene structure and gene order of the R. solani AG1-IB isolate7/3/14 mitochondrial genome feature specific characteristics

The number of protein coding genes is highly variable ineukaryotic mitochondrial genomes (Adams and Palmer, 2003).Fungal mitochondrial genomes include up to seven nad genes(NADH dehydrogenase subunits), a cob gene (cytochrome b),three cox genes (cytochrome c oxidase subunits), two to threeatp genes (ATP synthase F0 subunits), two ribosomal RNA genesand a ribosomal protein gene (Paquin et al., 1997). The R. solaniAG1-IB mitochondrial gene content is characteristic for fungalmitochondrial genomes. In addition to the core genes (see Table 5),R. solani AG1-IB mt-DNA comprises 12 long ORFs encodingunknown hypothetical proteins or endonucleases. Commonly,mitochondrial genes of Basidiomycota are located on one strand. Inthe R. solani AG1-IB 7/3/14 mitochondrial genome almost all genesare located on the clockwise strand. The only identifiable geneon the anti-clockwise strand encodes a tRNATrp. Likewise, thisorientation of a tRNA gene was also found in the Trametes cingulatamitochondrial genome (Haridas and Gantt, 2010). The gene order



Fig. 3. Example for local similarities between R. solani AG1-IB 7/3/14 group II contigs. This comparison exemplarily shows the two contigs 19,997 and 20,138 that share a20 kb long region with more than 94% sequence identity. The amount of mismatches was calculated on basis of non-overlapping intervals of the length 1000 bp. Numbers inboxes indicate mismatches in 1000 bp intervals. The results of each interval are summed up to 812 mismatches for the complete alignment.

within mt-DNA is not conserved among known Basidiomycotamitochondrial genomes. However, they share a similar set of genes.Only nad genes are mostly clustered (nad2–nad3, nad4L–nad5,and nad4–nad6) (Formighieri et al., 2008). In the R. solani AG1-IB7/3/14 mitochondrial genome only the nad genes nad2–nad3 andnad4L–nad5 are located in close proximity. Also the position, sizeand number of introns as well as genes with exon–intron structureare not conserved among known Basidiomycota mitochondrialgenomes. In the R. solani AG1-IB 7/3/14 mitochondrial genome,introns were detected in the atp6, cox1, cox2, cox3, cob, dpoB, nad1,nad2, nad4, nad5, nad6, and rnl genes (Table 5). Accordingly, theR. solani AG1-IB mitochondrial genome contains more multi-exongenes than other fungal mitochondrial genomes known to date.Splice sites of these genes are not conserved, but they mainly con-tain the bases T and G. All of the introns identified are type I introns,except for a single type II intron in the rnl gene. Commonly, typeI introns are characterized by the presence of long ORFs encoding

endonucleases. These endonucleases are involved in intron mobil-ity and are often referred to as homing endonucleases. They featurerare recognition sites and cleave the target gene. In this way, theDNA repair machinery of the cell is activated resulting in preciseinsertion (i.e. ‘homing’) of the intron sequence into the targetgene (Lang et al., 2007). Actually, all type I introns in the R. solani7/3/14 mitochondrial genome comprise endonuclease-like genesequences. The exon–intron structure of the cox1 gene is the mostcomplex in the R. solani AG1-IB mitochondrial genome. This is alsothe case in the mitochondrial genomes of the basidiomycete fungiT. cingulata, Pleuritus ostreatus, M. perniciosa, and Ustilago maydis.R. solani 7/3/14 intronic ORFs show higher sequence similarityto different Glomus strains (arbuscular mycorrhizal fungi) thanto closely related fungi. Likewise, comparison of the whole R.solani AG1-IB 7/3/14 mitochondrial genome with those of otherrelated Basidiomycota species did not show significant overallsynteny concerning protein coding genes. A DNA polymerase

Fig. 4. Physical map of the R. solani AG1-IB 7/3/14 mitochondrial genome. From the outer to the inner circle: (i) R. solani AG1-IB 7/3/14 CDSs (blue), tRNA genes (black) andrRNA genes (green) identified in the clockwise and anti-clockwise strand (the only gene in the anti-clockwise strand is the one encoding tRNATrp marked in yellow); (ii)exons in the clockwise strand; (iii) intronic ORFs in the clockwise strand; (iv) GC content as the deviation from the average over the entire genome and (v) GC skew as thedeviation from the average over the entire genome. The start of the cox1 gene has been defined as the first base of the mitochondrial genome.



Table 5Gene content and number of introns present in the mitochondrial genomes of thebasidiomycetes R. solani AG1-IB 7/3/14 (Rs), T. cingulata (Tc), Pleurotus ostreatus (Po),M. perniciosa (Mp), S. commune (Sc), Cryptococcus neoformans (Cn), U. maydis (Um),and the ascomycete Aspergillus niger (An) (according to Haridas and Gantt, 2010).

Genes Rs Tc Po Mp Sc Cn Um An

cox1 7 15 9 6 0 0 8 1cox2 2 2 1 2 0 0 0 0cox3 3 0 0 0 0 0 0 0atp6 1 0 0 0 0 0 0 0atp8 0 0 0 0 0 0 0 0atp9 0 0 0 0 0 0 0 0nad1 1 0 0 1 0 1 0 0nad2 1 0 1 0 0 0 0 0nad3 0 0 0 0 0 0 0 0nad4 1 0 1 1 0 0 0 0nad4L 0 0 0 0 0 0 0 1nad5 2 2 0 1 0 0 1 0nad6 1 0 0 0 0 0 0 0rnl 5 6 0 0 0 0 2 1rns 0 1 0 0 0 0 0 0rps3 0 0 0 0 0 0 0 –a

cob 2 0 0 2 0 0 1 0

a Absence of a gene is indicated by a dash (–). Gene names and correspondingfunctions are described in Section 3.6.

gene, a non-typical mitochondrial gene, that has been reportedfor P. ostreatus and M. perniciosa (Formighieri et al., 2008; Wanget al., 2008), is also present in the R. solani 7/3/14 mitochondrialgenome. Reported DNA polymerase genes encoded in the mito-chondrial genomes of P. ostreatus and M. perniciosa are supposedto originate from integrated plasmids. In summary, the R. solaniAG1-IB 7/3/14 mitochondrial genome encodes a core set of genesthat is also present in other mitochondrial genomes. However, thehigh number of multi-exon genes is characteristic for the R. solaniAG1-IB mt-genome. Moreover, bioinformatic and comparativeanalyses provided new insights into the mitochondrial genome ofR. solani AG1-IB 7/3/14. Structural divergence in mt-genomes hasbeen observed suggesting that different evolutionary forces wereinvolved in shaping and specification of mt-genomes. The R. solani7/3/14 mt-genome seems to possess a high plasticity as deducedfrom comparative analyses comprising other fungal mt-genomes.However, particular conserved characteristics within mt-genomesmost probably are attributed to their functional role.

3.7. Sequence and structure of the R. solani AG1-IB isolate 7/3/14region encoding ribosomal RNAs

Thirty-five contigs featuring a coverage higher than 50-foldcould be detected in the contig-length vs. read-count plot (Fig. 2).These contigs were compared to the NCBI database by means ofBLASTn indicating two groups of contigs: the first group includ-ing 23 contigs showed a high degree of similarity to ribosomalRNA genes. The remaining 12 contigs are short (<200 bp) and mostprobably represent highly repetitive microsatellite sequences.

To analyze the R. solani AG1-IB 7/3/14 rRNA unit in more detail,matching contigs were mapped to the complete rRNA unit of therelated Basidiomycota species Fibroporia vaillantii (accession no.M286436, Schmidt and Moreth, 2008), representing the most simi-lar and complete reference sequence. Mapping results revealed thatnine contigs nearly cover the complete rRNA unit (Fig. 5). The iden-tified R. solani AG1-IB 7/3/14 rRNA unit has a size of 9269 bp andconsists of the 18S rRNA gene, ITS1 region, 5.8S rRNA gene, ITS2region, 28S rRNA gene, IGS1 region, 5S rRNA gene and the IGS2region. Intergenic spacers between rRNA genes only show limitedsimilarity to the reference. However, the rRNA genes are highlyconserved in comparison to the F. vaillantii rRNA unit (18S: 93%,5.8S: 96%, 28S: 94%, 5S: 97%). The 18S, 5.8S, and 5S rRNA genes

only consist of one exon whereas the 28S rRNA gene is composedof three exons. Remaining contigs and contigs featuring lower cov-erage represent intra-genomic variations in the rRNA units. Similarobservations were described for rRNA gene regions of other fungi(Ganley and Kobayashi, 2007). Sequence variation occurs all overthe rRNA unit. However, as expected, non-essential parts of therRNA unit like the 28S rRNA gene introns, the ITS- and IGS-regionare less conserved. Sequence variation in rRNA units reflects thediploid and heterokaryotic character of the R. solani AG1-IB 7/3/14genome. All of the nine contigs representing the rRNA unit are morethan 100-fold over-represented in the genome indicating that R.solani AG1-IB 7/3/14 possesses multiple copies of the rRNA genecluster which has also been observed for other fungal genomes(Ganley and Kobayashi, 2007). Availability of the R. solani AG1-IB7/3/14 rRNA unit sequence offers the possibility to design specificprimers for detection of this isolate and for differentiation of mem-bers belonging to the R. solani species complex. For other isolatesand other AGs such specific primers were designed for this purpose(Godoy-Lutz et al., 2008; Salazar et al., 2000).

3.8. The R. solani AG1-IB 7/3/14 genome is only distantly relatedto other completely sequenced fungal genomes

Recently, the genome sequences of C. cinerea, L. bicolor, S. lary-mans, and S. commune became available. To determine the degreeof similarity to these genomes, the R. solani AG1-IB 7/3/14 draftgenome was analyzed by applying the bioinformatics tool r2cat(Husemann and Stoye, 2010). Moreover, the set of core scaffoldsshared by these genomes was estimated. The most closely relatedreference sequence for R. solani AG1-IB 7/3/14 is C. cinerea. The C.cinerea genome sequence consists of thirteen contigs represent-ing thirteen chromosomes and further 54 super-contigs. Results ofthe reference mapping to the C. cinerea draft genome are shown inFig. 6. Only few R. solani AG1-IB 7/3/14 scaffolds feature sequencesimilarity to this reference genome. Horizontal lines in the syntenyplot represent common microsatellite sequences that are presenton all C. cinerea chromosomes. Likewise, reference mappings to thegenomes of L. bicolor, S. larymans, and S. commune revealed limitedsimilarity. The nearly finished reference genomes are only distantlyrelated to the R. solani AG1-IB 7/3/14 draft genome sequence andhence cannot serve as appropriate references for comparative anal-yses.

3.9. Gene prediction and annotation provide first insights into theR. solani AG1-IB 7/3/14 gene structure and content

To gain first insights into the R. solani AG1-IB 7/3/14 gene struc-ture and content, gene prediction by applying the gene predictionprogram for eukaryotes Augustus (version 2.6) was accomplished.Pre-computed parameter sets for the two related Basidiomycotaspecies L. bicolor and C. cinerea were used to facilitate gene recogni-tion in the R. solani AG1-IB 7/3/14 genome. Gene predictions basedon these gene models showed differences: 15,929 genes werepredicted by applying the L. bicolor model, whereas 12,422 genesresulted in the approach using the C. cinerea model. To identifyunique predictions, i.e. genes that have no shared region withina gene predicted by the other approach, coding sequences wereanalyzed with the program cuffmerge from the cufflinks package.Almost all predicted loci from the approach applying the C. cinereamodel are contained in the L. bicolor set whereas the L. bicolorprediction comprises 3855 unique loci (see Table 6). To evaluatethe quality of the gene predictions, highly conserved glycolysisand citrate cycle enzymes from the SWISS-PROT database weresearched in the predictions. Both predictions revealed identically(28 proteins) or almost identically (11 proteins) predicted proteins.Based on this analysis the performance of the applied gene models



Fig. 5. Structure of the R. solani AG1-IB 7/3/14 rRNA unit. (A) Genes encoding 18S, 5.8S, 28S and 5S rRNAs are shown. (B) Exon–intron structure of genes. (C) Contigs coveringthe rRNA unit. (D) Additional contigs that were mapped to the rRNA unit.

cannot be distinguished. All fungal glycolysis and citrate cycleenzymes were detected in both gene predictions. However, dueto the more conservative gene prediction, further analyses werebased on genes derived from the approach applying the C. cinereaparameter set from Augustus.

The number of exons per gene ranges from one to up to 52exons with a mean value of 5.2 exons (Fig. 7A) which is in agree-ment with other sequenced species of the phylum Basidiomycota(Galagan et al., 2005). The exons have a mean length of almost214 bp ranging from 1 bp to 6598 bp (Fig. 7B). The introns have a

mean length of almost 66 bp, ranging from 33 bp to 948 bp (Fig. 7C).Using tRNAScan-SE, 167 tRNA genes for all amino acids were iden-tified.

To review taxonomic assignments of R. solani AG1-IB 7/3/14genes, best BLASTn hits against the nt database were determined.More than 45% of the R. solani AG1-IB 7/3/14 genes (5572 genes) didnot show significant similarity to sequences within the nt databaseindicating that database entries of closely related fungi are cur-rently missing in the nt database. Fig. 8 reflects the taxonomicclassification of the remaining genes (6850) for which homologous

Fig. 6. Mapping of R. solani AG1-IB 7/3/14 contigs to the C. cinerea okayama7#130 reference genome by means of the tool r2cat. Default settings were used for referencegenome mapping. Blue dots represent homologous regions between R. solani isolate 7/3/14 contigs and the C. cinerea okayama7#130 genome. Red areas in the bottom linerepresent non-homologous regions in the C. cinerea okayama7#130 genome. Gray vertical lines visualize the size of the different chromosomes in the reference sequence.



Fig. 7. Exon–Intron statistics of the R. solani AG1-IB 7/3/14 genome. (A) The distribution of exons per gene shows that the majority of genes possess 1–10 exons and onlyfew genes have more than ten exons. (B) The distribution of the exon lengths indicates that the majority of exons is shorter than 1 kb. Few exons are longer than 4 kb. (C)The mean length of introns is 70 bp and the intron length ranges from 25 bp to more than 40 kb.

Fig. 8. Hierarchical taxonomic assignments of R. solani AG1-IB 7/3/14 genes apply-ing BLASTn. The plot reflects the taxonomic assignments of genes deduced from bestBLASTn matches versus the nt database. The width of segments is proportional to thepercentage of genes with the corresponding taxonomic assignment. Numbers repre-sent the following taxonomic assignments: (1) Ascomycota 639, (2) Pucciniomycetes111, (3) Tremellomycetes 230, (4) Ustilaginomycetes 108, (5) Sordariomycetes 326, (6)Eurotiomycetes 157, (7) Polyporales 272, (8) Tremellales 224, (9) Ustilaginales 108, (10)Magnaporthales 197, (11) Eurotiales 114. Taxonomic assignments with less than 100hits were not considered.

sequences are present in the database. The majority of these genesare closely related to sequences originating from fungi, especiallyof those belonging to the class Agaricomycetes. Minor fractions ofR. solani AG1-IB 7/3/14 genes correspond to genes from more dis-tantly related fungi including those of the phylum Ascomycota, andthe classes Pucciomycetes, Tremellomycetes, and Ustilaginomycetes(Fig. 8). Interestingly, 87 genes could be classified to Bacteria and71 genes to eukaryotes of the kingdom Metazoa. In summary, insuf-ficient taxonomic classification of R. solani AG1-IB isolate 7/3/14genes indicates that genome sequences of Agaricomycetes classmembers are underrepresented or even missing in databases. As aconsequence, R. solani gene prediction results cannot be comparedto closely related species.

Table 6R. solani AG1-IB isolate 7/3/14 genes as predicted by applying the L. bicolor or C.cinerea parameter set within the gene prediction tool for eukaryotes Augustus.

Gene model deduced froma Total genes Unique genesb

C. cinerea 12,422 127L. bicolor 15,929 3855

a Gene prediction was done based on gene models deduced from the C. cinerea orL. bicolor genomes, respectively.

b Unique genes of one gene prediction applying a specific gene model in compar-ison to the second gene prediction applying the alternative gene model.



Table 7Most abundant (top 10) KOG categories of the predicted R. solani AG1-IB 7/3/14genes.

KOG-category Amountof hits

General function prediction only 1056Posttranslational modification, protein turnover, chaperones 393Energy production and conversion 285Signal transduction mechanisms 275Translation, ribosomal structure and biogenesis 262Secondary metabotites biosynthesis, transport and catabolism 257Intracellular trafficking, secretion, and vesicular transport 231Function unknown 217Amino acid transport and metabolism 196RNA processing and modification 193

To obtain functional information of the predicted genes, theywere functionally annotated using GenDB 2.0 (Meyer et al., 2003).KOG categories (eukaryotic ‘Cluster of Orthologous Groups of pro-teins’) and annotations were assigned to the genes based on theirbest BLAST hit by applying different databases (KEGG, SWISS-PROT, etc.). KOGs were delineated by comparing protein sequencesencoded in complete genomes, representing major phylogeneticlineages. Each KOG consists of individual proteins or groups oforthologous proteins from at least three lineages and thus cor-responds to an ancient conserved domain. R. solani 7/3/14 genesproduced hits to all 26 KOG functional categories. In total, KOGnumbers could be assigned to 4169 genes (Table 7). Due to the lackof closely related genome sequences in databases, functional inter-pretation of R. solani mainly has to be done by de novo analyses forthis species.

4. Concluding remarks

Analysis of fungal genomes by high-throughput sequencingtechnologies and new bioinformatics tools for sequence analysisand interpretation has added a new dimension to study evolution,taxonomy, gene content and life style of organisms belonging tothe important kingdom of fungi, especially of those entering intopathogenic interactions. Previously, different fungal genomes of thephylum Basidiomycota were completely sequenced and analyzed,e.g. C. cinerea, L. bicolor, S. larymans, and S. commune. However,these fungi are basically homokaryotic and their genomes couldbe sequenced in the haploid state.

In contrast, in this work, the first high quality draft genomesequence of the multinucleate and heterokaryotic fungus R. solaniAG1-IB 7/3/14 could be established for its diploid state. To reducethe relatively high number of contigs and scaffolds for the R. solaniAG1-IB 7/3/14 genome, an advanced third-generation sequenc-ing technology producing much longer read lengths (>3 kb) wouldbe required to facilitate scaffolding of contigs. Generation of ahomokaryotic R. solani derivative by means of bioengineeringmethods is not an option to improve the genome conciseness sincethis approach would reduce the genetic repertoire of the organ-ism and hence features and properties of such derivatives mostprobably would not be comparable to those of wild type isolates.Accordingly, the draft genome sequence established for a natural R.solani AG1-IB isolate provides its complete genetic layout. Furthersequencing of other R. solani AG1-IB isolates is required to estimatethe pan-genome and the overall degree of genome heterogeneityfor this important anastomosis group.

The primary gene prediction of R. solani isolate 7/3/14 will serveas a valuable resource for further analyses regarding pathogenicityof this fungus and identification of possible targets for biocontrolorganisms. Certainly, gene recognition in the R. solani AG1-IB 7/3/14genome still is insufficient since currently it is based on gene mod-els deduced from other sequenced Basidiomycetes. To overcome

this shortcoming, a R. solani gene model has to be developed andapplied in gene prediction for this important species. Results of thisstudy indicate that basic identification of genes within this group offungi seems to be quite consistent regarding loci representing coregenes of this phylum. However, experimental evidence regardinggene functions is still lacking in many cases. For R. solani, geneticengineering methods are insufficiently developed which preventsor at least complicates functional analyses. Validation of predictedgenes would require additional transcriptomic experiments suchas cDNA (Expressed Sequence Tag, EST) sequencing, RNA-Seq, ormicroarray hybridization. EST sequencing and RNA-Seq would beappropriate methods to validate not only the existence of pre-dicted genes, but also to identify the real intron-exon structuresof the genes. Development of a specific R. solani gene modeldeduced from mappings of EST data to genomic sequences wouldfacilitate recognition of so-far unknown genes including putativepathogenicity determinants of this species. In principle, pathogen-icity determinants can be detected in the completely annotated R.solani 7/3/14 genome in comparative analyses comprising relatedphytopathogenic fungi. Moreover, R. solani 7/3/14 transcriptomesequencing for a model system representing the pathogenic stateof the fungus may help to identify pathogenicity determinants. Cer-tainly, fully annotated and interpreted R. solani genomic resourcesare a prerequisite for improvements in disease control strategies.

Acknowledgments

L.J. and F.G.E. acknowledge the receipt of a scholarship fromthe CLIB Graduate Cluster ‘Industrial Biotechnology’ co-financedby the Ministry of Innovation of North Rhine-Westphalia. Thebioinformatics and technological support of the Bioinformatics andGenomics Platforms at the Center for Biotechnology (CeBiTec, Biele-feld University) is gratefully acknowledged. A.S. acknowledges theMETAEXPLORE grant of the European Commission (KBBE-222625).D.W., M.H. and A.S. thank the BMBF for funding of the PathControlcooperative project ‘Biological control of Bacillus amyloliquefacienson the soil-borne fungal pathogen R. solani (FKZ 0315654B).

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