Cruz Alta 16S rRNA soil metagenomic analysis
We analyzed soils from two agronomic sites and two non-agronomic sites according to the following gradient of soil disturbance in ascending order: Pristine (PR), Farmhouses yards (NA-FH), No-Till farming under good agronomical practices (NT-GP) and No-Till farming under poor agronomical practices (NT-PP). Sites were chosen within a short area to reduce the effect of geographic distances. The GPS coordinates to sampled sites were PR: - 32.94105, -61.844164; NA-FH: -32.856928, -62.007346; NT-GP: -32.85641,-62.007244 ;NT-PP: -32.940466,-61.845991. Soils in all sites are classified as typical Argiudolls with silty loam texture and good drainage (Panigatti 2010). Mean annual precipitation and temperature are 875 mm and 16.9 ºC respectively. The Pristine site is an extremely rare landscape in the region and is probably one of the few and last pieces of land that did not have any management for at least 200 years. The PR plot was covered by grasses and non-woody plants typical from the region that were probably dispersed by wind and animals. Farmhouse yards were few meters aside NT-GP and were planted with lawn. They have been used for this purpose for at least 20 years. Soils from NT-GP were managed under No-till farming the last 18 years using a wheat-soybean-corn rotation, alternating with periods of step-aside, fodder-plants and grasses and previously farmed for another at least 15 years with reduced tillage, growing mostly pastures and occasionally soybean. Soil chemical composition was often surveyed and nutrients were properly restored. Soils from NT-PP were managed under No-Till farming for at least the last 15 years, and farmed for more than 30 years. In this site, crop rotations were infrequent and were often managed with soybean monoculture. Nutrients were not properly restored after harvests. The last crop before sampling in both No-Till agronomic plots was soybean and samples were taken a month after harvest.     Samples were amplified in two rounds of PCR. First round was performed with 563F-802R universal primers (RDP Pyrosequencing Pipeline: http://pyro.cme.msu.edu/pyro/help) corresponding to the V4 hyper variable region of 16s rDNA gene fused at 5´end to a TAG sequence (TAG-fw: CACGACGTTGTAAAACGAC in primer forward and TAG-Rv: CAGGAAACAGCTATGACC in primer reverse). A second round of PCR was performed to add Roche 454 adaptors and a specific barcode to each sample. Forward primers used in second round were (from 5´to 3´): Roche 454 A adaptor, barcode, TAG-fw and Reverse primers were: Roche 454 B adaptor, TAG-rv. Two independent PCRs per sample were performed for first PCR round and products were pooled and used as template for two independent PCR reactions in the second round of PCR. In first amplification the PCR mix (final volume 25 ul) contained 2.5 ul FastStart High Fidelity 10X Reaction Buffer (Roche Applied Science, Mannheim, Germany), 20 ng of template DNA, 0.4 uM of each primer, 1.25 U FastStart High Fidelity Enzyme Blend (Roche Applied Science), and 0.2 mM DNTPs. The second round of PCR was made using the same mix but adding BSA (0.75 ul) and DMSO (0.8ul). The PCR conditions in the first amplification round were 95 °C for 5 minutes for initial denaturalization, followed by 95 °C for 45 seconds, 57 °C for 45 seconds, 72 °C for 60 seconds in 30 cycles, and a final elongation step at 72°C for 4 minutes. For the second round of PCR the program was 95 °C for 5 minutes for initial denaturalization, 20 cicles of 95 °C for 30 seconds, 72 °C for 60 seconds, and a final elongation step at 72°C for 5 minutes. -------------------------------------- QIIME Mapping file used to demultiplex samples:---------------------------------Replace @ for tab and $ for new line (\n)---------------------------------------------------------------------------------#SampleID@BarcodeSequence@LinkerPrimerSequence@ReversePrimer@PrimerType@Site@Pristine@Area@ANOVA@Box@Management@PlantType@Depth@LandUse@Description$ca.pNA.B.20.ind.a@TGATACGTCTCACGACGTTGTAAAACGAC@AYTGGGYDTAAAGNG@CAGGAAACAGCTATGACC@RDP@Pristine@Pristine@Cruz_Alta@PR@All_Other@NoAgro@Pristine@0-20cm@NA@ca.pNA.B.20.ind.a$ca.pNA.B.20.ind.b@CATAGTAGTGCACGACGTTGTAAAACGAC@AYTGGGYDTAAAGNG@CAGGAAACAGCTATGACC@RDP@Pristine@Pristine@Cruz_Alta@PR@All_Other@NoAgro@Pristine@0-20cm@NA@ca.pNA.B.20.ind.b$ca.pNA.B.5.comp.a@CGAGAGATACCACGACGTTGTAAAACGAC@AYTGGGYDTAAAGNG@CAGGAAACAGCTATGACC@RDP@Pristine@Pristine@Cruz_Alta@PR_sup@All_Other@NoAgro@Pristine@0-5cm@NA@ca.pNA.B.5.comp.a$ca.pNA.B.5.comp.b@ATACGACGTACACGACGTTGTAAAACGAC@AYTGGGYDTAAAGNG@CAGGAAACAGCTATGACC@RDP@Pristine@Pristine@Cruz_Alta@PR_sup@All_Other@NoAgro@Pristine@0-5cm@NA@ca.pNA.B.5.comp.b$ca.RP.NA.B.0y1.ind.a@TCACGTACTACACGACGTTGTAAAACGAC@AYTGGGYDTAAAGNG@CAGGAAACAGCTATGACC@RDP@RobertoP@RobertoP@Cruz_Alta@NA_0_1@NA_Sup.@NoAgro@Grass@0-1cm@NA@ca.RP.NA.B.0y1.ind.a$ca.RP.NA.B.0y1.ind.b@CGTCTAGTACCACGACGTTGTAAAACGAC@AYTGGGYDTAAAGNG@CAGGAAACAGCTATGACC@RDP@RobertoP@RobertoP@Cruz_Alta@NA_0_1@NA_Sup.@NoAgro@Grass@0-1cm@NA@ca.RP.NA.B.0y1.ind.b$ca.RP.SD.B.20.ind.a@TCTACGTAGCCACGACGTTGTAAAACGAC@AYTGGGYDTAAAGNG@CAGGAAACAGCTATGACC@RDP@RobertoP@RobertoP@Cruz_Alta@NT-GS@All_Other@Agro@None@0-20cm@NT@ca.RP.SD.B.20.ind.a$ca.RP.SD.B.20.ind.b@TGTACTACTCCACGACGTTGTAAAACGAC@AYTGGGYDTAAAGNG@CAGGAAACAGCTATGACC@RDP@RobertoP@RobertoP@Cruz_Alta@NT-GS@All_Other@Agro@None@0-20cm@NT@ca.RP.SD.B.20.ind.b$ca.RP.SD.B.0y1.ind.a@ACGACTACAGCACGACGTTGTAAAACGAC@AYTGGGYDTAAAGNG@CAGGAAACAGCTATGACC@RDP@RobertoP@RobertoP@Cruz_Alta@NT-GS_sup@All_Other@Agro@None@0-1cm@NT@ca.RP.SD.B.0y1.ind.a$ca.RP.SD.B.0y1.ind.b@CGTAGACTAGCACGACGTTGTAAAACGAC@AYTGGGYDTAAAGNG@CAGGAAACAGCTATGACC@RDP@RobertoP@RobertoP@Cruz_Alta@NT-GS_sup@All_Other@Agro@None@0-1cm@NT@ca.RP.SD.B.0y1.ind.b$ca.RP.SD.B.20.comp.a@TACGAGTATGCACGACGTTGTAAAACGAC@AYTGGGYDTAAAGNG@CAGGAAACAGCTATGACC@RDP@RobertoP@RobertoP@Cruz_Alta@NT-GS@All_Other@Agro@None@0-20cm@NT@ca.RP.SD.B.20.comp.a$ca.RP.SD.B.20.comp.b@TACTCTCGTGCACGACGTTGTAAAACGAC@AYTGGGYDTAAAGNG@CAGGAAACAGCTATGACC@RDP@RobertoP@RobertoP@Cruz_Alta@NT-GS@All_Other@Agro@None@0-20cm@NT@ca.RP.SD.B.20.comp.b$ca.RP.NA.B.20.comp.a@TAGAGACGAGCACGACGTTGTAAAACGAC@AYTGGGYDTAAAGNG@CAGGAAACAGCTATGACC@RDP@RobertoP@RobertoP@Cruz_Alta@NA-FH@All_Other@NoAgro@Grass@0-20cm@NA@ca.RP.NA.B.20.comp.a$ca.RP.NA.B.20.comp.b@TCGTCGCTCGCACGACGTTGTAAAACGAC@AYTGGGYDTAAAGNG@CAGGAAACAGCTATGACC@RDP@RobertoP@RobertoP@Cruz_Alta@NA-FH@All_Other@NoAgro@Grass@0-20cm@NA@ca.RP.NA.B.20.comp.b$ca.pSD.B.20.comp.a@ACATACGCGTCACGACGTTGTAAAACGAC@AYTGGGYDTAAAGNG@CAGGAAACAGCTATGACC@RDP@NearPristine@RobertoP@Cruz_Alta@NT-ST@All_Other@Agro@None@0-20cm@NT@ca.pSD.B.20.comp.a$ca.pSD.B.20.comp.b@ACGCGAGTATCACGACGTTGTAAAACGAC@AYTGGGYDTAAAGNG@CAGGAAACAGCTATGACC@RDP@NearPristine@RobertoP@Cruz_Alta@NT-ST@All_Other@Agro@None@0-20cm@NT@ca.pSD.B.20.comp.b$ca.pNA.B.20.comp.a@ACTACTATGTCACGACGTTGTAAAACGAC@AYTGGGYDTAAAGNG@CAGGAAACAGCTATGACC@RDP@Pristine@Pristine@Cruz_Alta@PR@All_Other@NoAgro@Pristine@0-20cm@NA@ca.pNA.B.20.comp.a$ca.pNA.B.20.comp.b@ACTGTACAGTCACGACGTTGTAAAACGAC@AYTGGGYDTAAAGNG@CAGGAAACAGCTATGACC@RDP@Pristine@Pristine@Cruz_Alta@PR@All_Other@NoAgro@Pristine@0-20cm@NA@ca.pNA.B.20.comp.b
CruzAlta