Chromosome size and number were analyzed for North American NFNB isolates; 0-1 and 15A, previously used to develop a genetic cross for identifying avirulence genes [39,63]. Fungal protoplasts were prepared using a protocol established for S. nodorum as described by Liu et al. [56] with some modifications. Briefly, conidia were harvested from 7-day fungal cultures and inoculated into 60 ml liquid Fries medium in 250 ml Erlenmeyer flasks. After growth at 27°C in a shaker (100 rpm) for 48 h, the fungal tissue was then homogenized in a Waring blender and re-inoculated into 200 ml liquid Fries medium in 500 ml Erlenmeyer flasks. The fungus was grown under the same growth conditions for 24 h. Mycelium was harvested by filtering through two layers of Miracloth, washed thoroughly with water and finally with mycelial wash solution (MWS: 0.7 M KCl and 10 mM CaCl₂). Around 2 g (wet weight) of mycelial tissue was then transferred into a Petri dish (100 × 20 mm) containing 40 ml filter-sterilized protoplasting solution containing 40 mg/ml β-d-glucanase, 0.8 mg/ml chitinase, and 5 mg/ml driselase (Interspex Product Inc., San Mateo, CA, USA) in MWS. The Petri dish was shaken at 70 rpm at 28°C for at least 5 h. Protoplasts were filtered through four layers of Miracloth and pelleted by centrifugation at 2,000 × g for 5 minutes at room temperature, followed by another wash with MWS and pelleting. Protoplasts were resuspended in MWS to a final concentration of 2 × 10⁸ protoplasts/ml and mixed with an equal volume of 2% low melting temperature agarose (Bio-Rad Laboratories, Hercules, CA, USA) dissolved in MWS. Agarose plugs were made by pipetting 80 μl of the mixture into plug molds (Bio-Rad Laboratories). Once solidified, plugs were placed in 20 ml Proteinase K reaction buffer containing 100 mM EDTA (pH 8.0), 1% N-lauroyl sarcosine, 0.2% sodium deoxycholate and 1 mg/ml Proteinase K (USBiological, Swampscott, MA, USA) at 50°C for 24 h. Plugs were washed four times in 10 mM Tris pH 8.0 and 50 mM EDTA for 1 h with gentle agitation, then stored in 0.5 M EDTA (pH 8.0) at 4°C. PFG was performed on a Bio-Rad CHEF Mapper system. Separation of chromosomes in the 1 to 6 Mb range was carried out in 1.0× TAE at 14°C using 0.8% Low EEO agarose gel (USBiological). Run time was 72 h at 2 V/cm (70 V) with a 20- to 40-minute switch time ramp at an angle of 106°.

DNA of P. teres f. teres isolate 0-1 was extracted using a Biosprint DNA Plant Kit and a BioSprint 15 automated workstation (Qiagen, Hilden, Germany). Genomic sequencing was performed on a Solexa sequencing platform at the Allan Wilson Centre (Massey University, Palmerston North, New Zealand). DNA preparation, cluster formation, primer hybridization and DNA amplification reactions were according to the manufacturer's recommended protocol [64]. DNA sequencing was performed using 75-bp paired-end reads of randomly sheared 200-bp fragments in a single flow cell. Data were pre-filtered in Illumina's Pipeline v.1.4 and IPAR v.1.3. Reads failing a 'chastity' filter of 0.6 were discarded. The steps described below for genome scaffold assembly, annotation and analysis were performed on the iVEC advanced computing facilities [65].

To validate the assembly over a larger scale, BLASTN [18] was used to compare the assembly against two NFNB 0-1 BACs, designated 8F17 and 1H13, sequenced and assembled by The Genome Center (Washington University, St Louis, MO, USA). The data were visualized with CIRCOS [69]. To establish if all regions of the BACs were covered by Solexa sequencing, raw reads were mapped to the BACs with the Burrows-Wheeler Aligner [70] and visualized using R and the ggplot2 package [66,71].