DNA extraction and sequencing procedures
DNA of the isolates from Japan and New Zealand, and the non-French
European isolates was extracted using a phenol-chloroform protocol
available at the JGI webpage (Isolation of genomic DNA fromPhytophthora :
http://jgi.doe.gov/collaborate-with-jgi/pmo-overview/protocols-sample-preparation-information/)
combined with the Qiagen Genomic tip protocol (Qiagen, Hilden, Germany).
Briefly, about 500 mg fresh mycelium, from isolates grown on Serpula
Capex Dox medium (see (Eastwood et al. 2011)), was flash frozen
in liquid N2 and ground to powder in a mortar. Sixty mL
extraction buffer (0.2 M Tris pH 8.5, 0.25 M NaCl, 25 mM EDTA, 0.5%
SDS) was added to the mortar before the sample was distributed into four
50 mL tubes. 15 mL 7:3 phenol:chloroform was added to each sample,
before they were mixed and incubated for 90 min at room temperature. The
samples were centrifuged at 6000 G for 15 min. The aqueous phase was
added to equal volume (V) of chloroform. After mixing, the samples were
centrifuged for another 5 min on 6000 G. The aqueous phase was
transferred to a clean tube and 0.6 V isopropanol added. The samples
were incubated on ice for 30 min before centrifugation at 4 °C at 6000 G
for 15 min. The pellet was washed with 20 mL 70% EtOH and dried for
five min at RT. The DNA was then dissolved in 500 μL of milli-Q
H2O. The four samples were pooled, 2 mg RNase A
(Invitrogen) was added, and incubated for 90 min at 37 °C. Eight mL
Qiagen QBT buffer was added, and the mix transferred to Genomic-tip
columns and processed following the Qiagen Genomic-tip protocol. The
three samples SL200, SL198 and SL265 were extracted without using the
Genomic-tip protocol, according to Balasundaram et al . (2018).
DNA of the French isolates was extracted using a CTAB – Qiagen genomic
tip protocol as described in Payen et al . (2015).
Genomic DNA was used to construct paired-end (2 x 125 bp) libraries
using SBS v4 Sample Prep Kit. Libraries were sequenced using the
Illumina HiSeq 2500 platform (Illumina, Inc., San Diego, CA, USA) at the
GeT-PlaGe sequencing facility (Toulouse, France). For the isolates
SL200, SL198 and SL265 (2 x 108 bp) libraries were sequenced on the
Illumina GAII at the SNP&SEQ Technology Platform (Uppsala, Sweden).