North2south biotin random prime labeling kit

Fungal genomic DNA was extracted with a cetyltrimethylammonium bromide protocol according to The Fusarium Laboratory Manual (94 (link)). Restriction endonuclease digestion, agarose gel electrophoresis, and gel blotting were performed following standard protocols (95 (link)). A North2South Biotin Random Prime Labeling Kit and a Chemiluminescent Hybridization and Detection Kit (Thermo Scientific, USA) were used for Southern blot hybridization. Total RNA was extracted using an Easy-Spin Total RNA Extraction Kit (Intron Biotech, Korea).

The genomic DNA was extracted as previously described (1 ). Total RNA was extracted from mycelia ground in liquid nitrogen using an Easy-Spin Total RNA Extraction Kit (Intron Biotech, Seongnam, Republic of Korea). Standard protocols were used for the restriction endonuclease digestion and agarose gel electrophoresis (72 ). Southern blot hybridization was performed using a North2South Biotin Random Prime Labeling Kit and a North2South Chemiluminescent Hybridization and Detection Kit (Thermo Scientific, USA) or by following standard techniques (72 ). The PCR primers (Table S2) were synthesized by an oligonucleotide synthesis facility (Bionics, Seoul, Republic of Korea).

Fungal genomic DNA was extracted with a cetyltrimethylammonium bromide protocol according to The Fusarium Laboratory Manual (94 (link)). Restriction endonuclease digestion, agarose gel electrophoresis, and gel blotting were performed following standard protocols (95 (link)). A North2South Biotin Random Prime Labeling Kit and a Chemiluminescent Hybridization and Detection Kit (Thermo Scientific, USA) were used for Southern blot hybridization. Total RNA was extracted using an Easy-Spin Total RNA Extraction Kit (Intron Biotech, Korea).

Genomic DNA of L. incisa was double digested with XhoI/NotI or HindIII/NotI restriction endonucleases at 37°C for 4-6 h. The digested DNA samples were fractionated on a 1.0% agarose gel and then transferred to a NC filter membrane (Millipore). A pair of primers was designed based on the conserved domain of GPAT (Supplementary Table S1). A 311-bp biotin-labeled DNA sequence was prepared to use as a probe with a North2South^® Biotin Random Prime Labeling Kit (Thermo Scientific). Subsequently, the hybridization was detected by the standard Southern blot procedure (Sambrook and Russell, 2001 ) with a North2South Chemiluminescent Hybridization and Detection Kit (Thermo Scientific). Signals were visualized by exposure to XBT-1 film (Kodak) at room temperature for 60-120 s.

The G418-resistant colonies were picked from the agar medium with 20 µg/ml G418, and cultured in 96-well plates containing modified CSi medium without G418 for 2 weeks, followed by scaling-up to 10 ml. Genomic DNA was extracted from the cultured cells using NucleoSpin Tissue XS (Takara Bio Inc., Shiga, Japan) or NucleoBond Buffer Set III and Nucleo BondAXG100 (Takara Bio Inc., Shiga, Japan). Insertion of nptII was first confirmed by PCR with the primer set 5′-ATG ATT GAA CAA GAT GGA TTG C-3′ and 5′-TCA GAA CTC GTC AAG AAG G-3′. Subsequently, 10 µg of genomic DNA was digested using restriction enzymes BglII and SalI. The digested DNA was subjected to electrophoresis using a 0.8% agarose gel, and transferred to Amersham Hybond-N+ membrane (GE Healthcare UK Ltd., Buckinghamshire, UK) using 20× saline sodium citrate (SSC) buffer, followed by depurination, denaturation and neutralization. Biotin-labelled probe for hybridization was synthesized using the total length of nptII fragment (795 bp) amplified by PCR and North2South Biotin Random Prime Labeling kit (Thermo Fisher Scientific Inc., Waltham, MA). The probe hybridizing with the genomic DNA on the membrane was detected using North2South Chemiluminescent Hybridization and Detection kit (Thermo Fisher Scientific Inc.), and C-Digit Blot Scanner (LI-COR Biosciences Inc., Lincoln, NE, USA).