16S rRNA seq of barley rhizosphere: mutant1 tp1
DNA was extracted from 0.25 g of rhizosphere soil using the PowerSoil DNA Isolation Kit (QIAGEN, USA), following the manufacturers protocol. To determine the bacterial community composition, Illumina Miseq sequencing was performed. The libraries of bacterial DNA were generated by a two-step dual-indexing PCR strategy, as previously described (Kudjordjie et al., 2019). V3/V4 region of 16S ribosomal RNA (rRNA) was amplified using primers 341F (S-DBact-0341-b-S-17: 5-CCTACGGGNGGCWGCAG-3) and 785R (S-D-Bact-0785-a-A-21: 5-GACTACHVGGGTATCTAATCC-3) (Klindworth et al., 2013). PCR amplifications were performed in a 25 L reaction mixture, using 1PCR buffer (Promega Corporation, Madison, WI, USA), 1.5 mM MgCl2, 0.2 mM dNTPs, 1 M each forward and reverse primer, 1 U of GoTaq Flexi polymerase (Promega Corporation), and 1 l of DNA template. The PCR program was as follows: 25 cycles of 94C for 30 s, 55C for 30 s, and 72C for 30 s, and final elongation at 72C for 10 min. Subsequently, a 15-cycle indexing PCR was performed with a similar PCR mixture and program as described above. A dual-indexing approach was employed for index combinations (Siddique & Unterseher, 2016; Wu et al., 2015), utilizing primer sequences with internal barcodes and indexes previously reported by Kudjordjie et al. (2019). To confirm the amplicon size of PCR products, SYBR staining was used in a 1.5% agarose gel for visualization. The barcoded PCR products were equimolarly pooled for equal representation, followed by ethanol precipitation and resolution in 50l of Tris-EDTA buffer. Subsequently, the pooled DNA was separated on a 1.5% agarose gel, and the DNA band of the expected size was extracted using a QIAquick Gel Extraction Kit (Qiagen). Afterward, the concentration of the extracted product was determined using a Nanodrop 1000 spectrophotometer. In the end, the amplicon sequencing was performed on a MiSeq platform by Eurofins MWG (Ebersberg, Germany).