Genetic Diversity and Abundance of Nitrogenase (nifH) and 16S rDNA Gene in the       Phyllosphere Samples of an Orchard in North China
16S rDNA were amplified using specific primers         515F(5’-GTGCCAGCMGCCGCGG-3’) and 907R (5’-CCGTCAATTCMTTTRAGTTT-3’), targeting the V4-V5         hypervariable regions (392bp)[49]. PCR was carried out with the following cycle conditions:         denaturation at 95 °C for 3 min, 27 cycles of 30 s at 95 °C, 30 s at 55 °C and 45 s at 72         °C, then 72 °C for 10 min. The reaction system was composed of 4 µL 5×FastPfu Buffer, 2 µL         2.5 mM dNTPs, 0.8 µL of both forward and reverse primer, 0.4 µL FastPfu Polymerase, 10 ng         template and Nuclease-Free Water to a final volume of 20 µL. Amplicons were purified using         AxyPrepDNA Gel Extraction Kit (AXYGEN, USA), and quantified using a fluorometric kit         (QuantIT PicoGreen, Invitrogen). The purified amplicons were then pooled in equimolar and         subject to Illumuna MiSeq sequencing.