Epiphytic community of olive tree fruits
The DNA was amplified for the hypervariable region with specific primers and further reamplified in a limited-cycle PCR reaction to add sequencing adapters and dual indexes. First PCR reactions were performed for each sample using KAPA HiFi HotStart PCR Kit according to manufacturer suggestions, 0.3 M of each PCR primer: a pool of forward primers ITS3NGS1_F 5-CATCGATGAAGAACGCAG-3, ITS3NGS2_F 5-CAACGATGAAGAACGCAG-3, ITS3NGS3_F 5-CACCGATGAAGAACGCAG-3, ITS3NGS4_F 5-CATCGATGAAGAACGTAG-3, ITS3NGS5_F 5-CATCGATGAAGAACGTGG-3, and ITS3NGS10_F 5-CATCGATGAAGAACGCTG-3 and reverse primer ITS4NGS001_R 5-TCCTSCGCTTATTGATATGC-3 (Tedersoo et al., 2014) and 12.5 ng of template DNA in a total volume of 25 L. The PCR conditions of involved a 3 min denaturation at 95 C, followed by 35 cycles of 98 C for 20 s, 58 C for 30 s and 72 C for 30 s and a final extension at 72 C for 5 min. Second PCR reactions added indexes and sequencing adapters to both ends of the amplified target region according to manufacturers recommendations (Illumina, 2013). Negative PCR controls were included for all amplification procedures. PCR products were then one-step purified and normalized using SequalPrep 96-well plate kit (ThermoFisher Scientific, Waltham, USA) (Comeau, Douglas, & Langille, 2017), pooled and pair-end sequenced in the Illumina MiSeq sequencer with the V3 chemistry, according to manufacturers instructions (Illumina, San Diego, CA, USA) at Genoinseq (Cantanhede, Portugal).