Root associated fungi of Bistorta vivipara
Samples were prepared for 454 pyrosequencing by performing nested PCR amplification using the fungal specific primers ITS1F and ITS4 (Gardes & Bruns 1993; White et al. 1990) in the first step, and fusion primers including ITS5 and ITS2(White et al. 1990) in the nested step. Fusion primers were constructed by adding five different 6 basepairs unique tags and 454 pyrosequencing adaptors A and B to both ITS5 and ITS2,  respectively. Tags were at least 3 bp different from each other in both directions. PCR was performed in 20 µL reaction volumes containing 2 µL template DNA and 18 µL reaction mix. Final concentrations in the PCR reactions were 0.16 mM dNTP mix, 0.2 µM of each primer and 0.4 units Finnzymes Phusion polymerase. The amplification program for both steps of the nested PCR was as follows: 30 s at 98ºC, followed by 30 cycles of 10 s at 98ºC, 20 s at 55ºC, 20 s at 72ºC, and a final extension step at 72ºC for 7 min before storage at 4 ºC. PCR products were cleaned with Wizard® SV Gel and PCR Clean-Up System (Promega, Madison, USA), quantified  using a bioanalyzer (Algilent 2100), and pooled into 12 equimolar amplicon libraries.