title: Microbial 16S rRNA gene-based composition of a sorghum cropped rhizosphere soil under different fertilization managements
design: The study focus on five managements encompassing four traditionally plowed and irrigated sorghum cropped plots under different N fertilizer amendments and an unmanaged grassland soil covered by spontaneous vegetation (GS). The four managed soils were: chemical fertilized soil by 130 kg of urea-N ha-1 (CT), compost amended soil by 130 kg of N ha-1 (COM1), compost amended soil by 260 kg of N ha-1 (COM2), and an unfertilized soil (UF). The experimental design utilized 5 m x 8 m plots and was a completely randomized block with four replicates (a, b, c, d) for each soil treatment.  Total community DNA was extracted from 0.5 g of soil using the FastDNA®SPIN Kit for Soil (MP Biomedicals, LLC).   For pyrosequencing, partial bacterial 16S rRNA gene fragments were PCR-amplified in 20-cycle reactions using primer 515R-NK linked to the 454 Life Science’s FLX-A sequencing adaptor, and a sevenfold-degenerate primer 27F-YM+3 linked to the FLX-B sequencing adaptor. An 8-nt sample-specific barcode was added between the A-adaptor and primer 515R-NK and was used to bioinformatically separate amplicons from the multiplexed reactions post-sequencing.     Each DNA was separately amplified in a 25 μl PCR reaction that contained 1.25 μl (20-50 ng) of DNA, 12.5 pmol of each primer, and 22.5 μl of Platinum® PCR SuperMix High Fidelity (Invitrogen). Reaction conditions included an initial denaturation at 95°C for 3 min followed by 20 or 30 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 30 s, and extension at 68°C for 1 min; and a final elongation at 68°C for 4 min on a Veriti® 96-well Thermal Cycler (Applied Biosystems). The PCR products were electrophoresed on 1% agarose gel in 0.5x TBE, stained with SYBR® Green I nucleic acid gel stain (Invitrogen) and scanned using Typhoon Trio+ Variable mode imager (GE Healthcare). The resultant images were quantified using Image Quant 5.2 (Molecular Dynamics). PCR products from replicates of each soil sample were then pooled in equimolar concentrations, followed by gel elution using Zymoclean™ Gel DNA Recovery Kit (Zymo Research). The eluted amplicons were quantified on the Experion System (Bio-Rad), followed by mixing of amplicons from all soil ages in equimolar concentration. The final mixed amplicon pool was further purified using the Agencourt AMPure XP system (Beckman Coulter Genomics) and submitted to the Georgia Genomics Facility at the University of Georgia (Athens, USA) for pyrosequencing. Emulsion PCRs were carried out as per their protocols.     Forward Sequecing primer: CCGCNGCKGCTGGCAC.    The table below describes the barcodes for the samples.       Sample   Library Barcode  CT TRA3A, rep 1 AACCGCAT  CT TRA3B, rep 2 AACCGCTA  CT TRA3C, rep 3 AACCTTCC  CT TRA3D, rep 4 AAGGAACC  COM1 CPT13A, rep 1 AAGGTTCC  COM1 CPT13B, rep 2 ACACACAC  COM1 CPT13C, rep 3 ACACCTCT  COM1 CPT13D, rep 4 ACACCTGA  COM2 CPT23A, rep 1 ACACGACT  COM2 CPT23B, rep 2 ACACGAGA  COM2 CPT23C, rep 3 ACACTCTC  COM2 CPT23D, rep 4 ACACTGAC  UF ON3A, rep 1 ACCTACCT  UF ON3B, rep 2 ACCTACGA  UF ON3C, rep 3 ACCTCATC  UF ON3D, rep 4 ACCTCTAC  GS IN3A, rep 1 ACCTGAAC  GS IN3B, rep 2 ACCTGTTC  GS IN3C, rep 3 ACCTTCGT  GS IN3D, rep 4 ACCTTGGA