RNA sequencing and variant discovery in the ash dieback pathogen (Hymenoscyphus fraxineus), using several infected samples collected from woodlands of Norfolk, UK

Dieback of Ash (Fraxinus excelsior L.) is caused by Hymenoscyphus fraxineus (previously known as H. pseudoalbidus; anamorph Chalara fraxinea) and widely reported in many sites in the UK. Ash trees are predominantly planted for forestry purposes and they make up 5.4% of British woodlands. H. fraxineus is a recent introduction into Europe from Asia. In a collaboration with colleagues across Europe, the NORNEX consortium aims to acquire rapid understanding of the pathogenicity of the fungus and genetics of resistance in the host. A single C. fraxinea isolate was cultured from infected tissue found in Kenninghall Wood (KW), Norfolk, UK. Genome sequence was assembled from the paired-end DNA libraries constructed and sequenced on an Illumina MiSeq sequencer for KW1 strain. The genome sequence data were produced in a coordinated effort between The John Innes Centre (JIC), The Sainsbury Laboratory (TSL) and The Genome Analysis Centre (TGAC) in Norwich. In the present study, we have collected 6 infected samples from different woodlands in Norfolk, UK. Fungal infected ash branches were collected from following locations; 2 samples were collected from Ashwellthorpe Lower Wood (AT), where Ash dieback was first reported in UK - AT1 and AT2 and 1 sample each from Upton broad and marshes (UB1); Foxley wood (FW1); Lolly moor (LM1) and Wayland wood (WW1). RNA was extracted from the pith material of the infected ash branches and were subjected to paired end RNA-Seq using Illumina GAII. Resulting sequence libraries were used to generate variant information for the 6 fungal samples against KW1 version 1 reference sequence.