ITS gene sequencing Vitis vinifera: leaves
The ITS1 region of the fungal ITS rDNA gene (Schoch et al. 2019) was amplified using primers ITS1F-ITS2 (White et al., 1990; Gardes and Bruns, 1993). To avoid a two-stage PCR protocol, each primer contained the Illumina adaptor sequence and a tag (ITS1F: 5- CAAGCAGAAGACGGCATACGAGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTxxxxxxxxxxxxCTTGGTCATTTAGAGGAAGTAA-3; ITS2: 5- AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTxxxxxxxxxxxxGCTGCGTTCTTCATCGATGC-3, where x is the 12 nucleotides tag). The PCR mixture (20 L of final volume) consisted of 10 L of 2X QIAGEN Multiplex PCR Master Mix (2X final), 2L each of the forward and reverse primers (0,1 M final), 4 l of water, 1 L of 10mg.ml-1 BSA and 1 L of DNA template. PCR cycling reactions were conducted on a Veriti 96-well Thermal Cycler (Applied Biosystems) using the following conditions: initial denaturation at 95 C for 15 min followed by 35 cycles at 94C for 30 s, 57C for 90 s, 72C for 90 s with final extension of 72 C for 10 min. ITS1 amplification was confirmed by electrophoresis on a 2% agarose gel.