Soil sample under wheat-maize crop rotation, conventional tillage and crop residue removal, plot 22, 18S rRNA
Metagenomic DNA was extracted from soil samples using the Power Soil DNA Isolation Kit (MO BIO Laboratories, CA, USA) following the manufacturer’s instructions. The V1–V3 region (about 550 bp) of archaeal 16S rRNA gene was chosen for amplification and subsequent pyrosequencing. The DNA samples were amplified using the set of primers for microscopic eukaryotes nu-SSU-0817 and nu-SSU-1196 (Rousk et al., 2010). Each ribosomal primer set was flanked by a 454–adapter sequence. A 10–nucleotide tag was incorporated between the 454–adapter and the forward primer for sample identification among mixed amplicon libraries. PCR products per soil sample were amplified in triplicate with a 30 cycle–based protocol, pooled and purified using the DNA Clean and Concentrator™ Columns (Zymo Research, Irvine, CA, USA).