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Abstract

Improved genome sequence of the phytopathogenic fungus Rhizoctonia solani AG1-IB 7/3/14 as established by deep mate-pair sequencing on the MiSeq (Illumina) system.

The fungus Rhizoctonia solani Kühn (teleomorph Thanatephorus cucumeris [Frank] Donk) belongs to the phylum Basidiomycota and is a worldwide prevalent soilborne plant pathogen which causes diseases in many economically important crops ( González García et al., 2006). R. solani is a species complex of distinct genetic groups named anastomosis groups (AGs). Currently, 13 AGs are known for R. solani featuring defined degrees of host specificity ( Carling et al., 2002). Draft genome sequences are available for four R. solani isolates representing three of the 13 AGs. These are the genomes of the rice sheath blight pathogen R. solani AG1-IA ( Zheng et al., 2013), an AG3 isolate, that is pathogenic on agricultural crops belonging to the plant family Solanaceae ( Cubeta et al., 2014), an AG8 isolate, representing a severely harmful pathogen causing bare patch of cereals, Brassica species and legumes ( Hane et al., 2014), and R. solani AG1-IB (isolate 7/3/14) ( Wibberg et al., 2014a and Wibberg et al., 2013), an isolate that was shown to be highly aggressive on lettuce and other vegetable crops such as bean, cabbage and carrot ( Grosch et al., 2007 and Grosch et al., 2004). The previous R. solani AG1-IB (isolate 7/3/14) genome and transcriptome sequences were established on the Genome Sequencer (GS) FLX platform ( Wibberg et al., 2014a and Wibberg et al., 2013). Genome assembly for this isolate resulted in a high number of contigs and scaffolds, also in comparison with other sequenced R. solani isolates, suggesting that intrinsic characteristics of the R. solani AG1-IB genome, e.g. repetitive elements, prevent further joining of contigs in assemblies. R. solani AG1-IB transcriptome analyses revealed Expressed Sequence Tags (ESTs) predicted to encode putative cellulose, pectin and lignin degrading enzymes that potentially can be exploited for biotechnological processes ( Wibberg et al., 2014a). Previously, high-throughput sequencing of mate-pair libraries has been established on the Illumina MiSeq system to achieve high quality genome sequences for different bacterial and archaeal species ( Hahnke et al., 2015, Koeck et al., 2014, Maus et al., 2013, Tomazetto et al., 2014 and Wibberg et al., 2014b).

In this study, the R. solani AG1-IB isolate 7/3/14 was resequenced to improve its genome sequence by means of deep sequencing on the Illumina MiSeq system. Compared to the GS FLX sequence data from the previous approach, sequence reads obtained on the MiSeq system provide a higher accuracy and better resolution of larger homopolymer stretches. Moreover, for resequencing an 8-k mate-pair library was generated that should improve contig scaffolding in assemblies in comparison to the 3-k paired-end library of the first sequencing approach.

The MiSeq sequencing run (2 × 250 bp) resulted in 11,603,267 processed reads yielding approx. 2.0 Giga bases sequence information. A de novo assembly of the obtained Illumina sequence reads by means of the Newbler Assembly software (version 2.8.) applying the heterozygotic mode generated 28,500 contigs accounting for a total length of 47.5 Mb ( Table 1). These assembly results are comparable to those achieved for the previous sequencing approach on the GS FLX system ( Wibberg et al., 2013). The MiSeq-based R. solani AG1-IB 7/3/14 draft genome sequences features a GC-content of 48.1%, which is identical to the GC-content of the first genome version.



Wibberg, D.; Rupp, O.; Jelonek, L.; Kröber, M.; Verwaaijen, B.; Blom, J.; Winkler, A.; Goesmann, A.; Grosch, R.; Pühler, A.; Schlüter, A. 2015. Improved genome sequence of the phytopathogenic fungus Rhizoctonia solani AG1-IB 7/3/14 as established by deep mate-pair sequencing on the MiSeq (Illumina) system. Journal of Biotechnology 203, 19-21.