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Supplementary Material
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The bacterial 16S rRNA gene and the fungal ITS1 region were amplified from the total DNA using FAM
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(6-carboxyfluorescein)-labeled polymerase chain reaction (PCR) forward primer and an unlabeled
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reverse primer. The primers used for the 16S rRNA gene and the ITS1 region were B27-FAM (5'-
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AGAGTTTGATCCTGGCTCAG) (Lane 1991), 1401-R (5'- CGGTGTGTACAAGACCC) (Nübel et al. 1996), ITS1F-
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FAM
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TCCTCCGCTTATTGATATGC) (White et al. 1990), respectively. For the 16S rRNA gene, the 50 µl reaction
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mixture contained 5 µl of 10x PCR buffer, 2.5 µl MgCl2 50 mM, 5 µl dNTPs 2 mM, 1 µl of each primer,
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1.25 µl DMSO, 1 µl BSA 3%, 500 U Taq-Polymerase, 30.75 µl H2O (DEPC), and 2 µl DNA from each
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sample. Amplification of the 16S rRNA gene was performed in the T3 Thermocycler (Biometra, Germany)
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using the following program: a 5 min hot start at 94ºC, followed by 35 cycles consisting of denaturation
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(1 min at 94ºC), annealing (1 min at 57ºC), extension (1 min at 72ºC), and a final extension step for 10
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min at 72ºC. For amplifying the fungal ITS1 region, the 100 µl reaction mixture contained 10 µl of 10x
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PCR buffer, 10 µl of 10x Corralload, 5 µl MgCl2 25 mM, 10 µl 5x Q-Solution, 0.5 µl Top Taq DNA
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polymerase (Top Taq DNA Polymerase, Qiagen, Germany), 10 µl dNTPs 2 mM, 48.5 µl H2O (DEPC), 2 µl of
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each primer, and 2 µl DNA of each sample. Amplification of the ITS1 region was performed on the T3
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Thermocycler (Biometra, Germany) with the following program: a 5 min hot start at 94ºC, followed by 35
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cycles consisting of denaturation (1 min at 94ºC), annealing (1 min at 50ºC), extension (1 min and 30s at
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72ºC), and a final extension step at 72ºC for 10 min.
(5’-
CTTGGTCATTTAGAGGAAGTAA)
(Gardes
and
Bruns
1993),
and
ITS4
(5’-
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T-RFLP analysis
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Diversity analysis by terminal restriction fragment length polymorphism (t-RFLP) was also performed
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targeting the bacterial 16S rRNA gene and the fungal ITS 1 region. For amplification, primer pairs and
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PCR profiles were performed, as described for PCR. Purification of the PCR products was performed with
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the NucleoSpin Gel and PCR clean-up kit (Macherey-Nagel, Germany), and digestion was performed
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using the restriction enzyme MSP1 (New England Biolabs, Germany) (Sakamoto et al. 2004) for the 16S
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rRNA gene, and Hinf1 (Fermentas, Lithuania) (Koide et al. 2007) for the ITS1 region. For digestion with
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MSP1, one microliter of the enzyme, 5 µl of 10x buffer, 10 µl H2O (DEPC), and 200 ng of the amplicon
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were used by using following program on the thermocycler: 4 h at 37ºC and 20 min at 80ºC. For
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digestion with Hinf1, 0.5 µl of the enzyme, 2.5 µl buffer, 12 µl H2O (DEPC), and 200 ng of the amplicon
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were used by using the following program on the thermocycler: 3 h at 37ºC and 20 min at 65ºC. The
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digested amplicons (50 ng) were desalted and purified with the Gel and PCR clean-up kit (Macherey-
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Nagel). One microliter was then mixed with 13 μl of Hi-Di formamide (Applied Biosystems, Germany)
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containing a 400-fold dilution of a 6-carboxy-X-rhodamine-labeled MapMarker 1000 ladder (Bio-
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Ventures, USA), denatured (5 min at 95ºC), cooled on ice, and size-separated on a 3730 DNA analyzer
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(Applied Biosystems). Electrophoresis was performed with POP-7 polymer in a 50-cm capillary array
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under the following conditions: 10 s injection time, 2 kV injection voltage, 7 kV run voltage, 66ºC run
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temperature, and 63 min analysis time. Electropherograms were analyzed using the GeneMapper 3.5
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software package (Applied Biosystems).
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