Genomic DNA extraction for re-sequencing of P. oryzaeisolates
To extract genomic DNA matching the quality criteria for full genome
sequencing, the 46 P. oryzae isolates selected above were first
grown on rice flour solid medium for mycelium regeneration, then in
liquid rice flour medium following
(2007). Genomic DNA
extraction was carried out using 100 mg of fresh mycelium from liquid
culture. Fresh mycelium dried on Miracloth paper was crushed in liquid
nitrogen. Nucleic acids were subsequently extracted with a lysis buffer
(2 % CTAB - 1.4 M NaCl - 0.1 M Tris-HCl pH 8 - 20 mM EDTA pH 8 added
before use with 1 % final of Na2SO3),
then purified with a chloroform:isoamyl alcohol 24:1 treatment,
precipited overnight in isopropanol, and rinsed with 70% ethanol. The
extracted nucleic acids were further treated with RNase A (0.2mg/mL
final) to remove RNA and purified with another chloroform:isoamyl
alcohol 24:1 treatment followed by an overnight ethanol precipitation.
The concentration of extracted genomic DNA was assessed on Qubit® using
the dsDNA HS Assay Kit. The purity of extracted DNA was checked by
verifying that the 260/280 and 260/230 absorbance ratios measured with
NanoDrop were between 1.8 and 2.0. We also ran 0.5 ot 1 µg DNA extracts
on agarose gel to visually verify the absence of RNAs and degraded DNAs.
Preparation of sequencing libraries and Illumina HiSeq 2500 sequencing
was performed at GenWiz Inc. USA, resulting in paired-end reads of 150
nucleotides with ca. 500 bp insert size.