ITS Amplicon seq of Artemisia species
DNA was extracted using the DNeasy Plant Pro Kit (QIAGEN, Germany), following the manufacturers instructions. For bulk and rhizospheric soils, 250 mg of soil was processed using DNA extraction with the DNeasy PowerSoil Pro Kit (QIAGEN, Germany). DNA concentrations were assessed with a BioSpectrometer (Eppendorf, Germany), and integrity was confirmed by electrophoresis on a 1% (w/v) agarose gel and visualized under UV light. Extracted DNA was stored at -20C. To minimize amplification of chloroplast DNA, fungal ITS2 regions were amplified using the primers with Custom Sequence (CS) adapters: ITS3 (TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAHCGATGAAGAACRYAG), and ITS4 (GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTCCTCCGCTTATTGATATGC) . PCR reactions were performed in a final volume of 25L containing 12.5L Platinum Direct PCR Universal Master Mix (ThermoFisher, Temara, Morocco). Thermal cycling conditions were: 94 C for 2min, 40cycles of 94 C for 15s, 60 C for 15s and 68 C for 20 sec; and a final extension at 72 C for 1min. Amplifications were performed on a Mastercycler X50s (Eppendorf, Germany). Amplicons were verified on 1% agarose gels visualized with iBright FL1500 Imaging System (ThermoFisher, Temara, Morocco). A non-template controls (NTC) were included in each run.