Uncultured root-associated fungi from Colorado
Root samples were collected from 60 individual plants (10 samples per species per habitat). Total DNA was extracted from each root sample and eluted in 100 µl of the EB elution buffer using Qiagen Plant DNeasy kits. Fungal small subunit (SSU) ribosomal DNA sequences were amplified using primer constructs that combined the A and B primers for 454 pyrosequencing with the fungus-specific SSU primers nu-SSU-0817-5' and nu-SSU-1536-3' (Borneman and Hartin, 2000). A 5 base pair (bp) DNA tag was inserted between the A and nu-SSU-1536-3' primer for post-sequencing sample identification. Each template was PCR-amplified in three separate 20 micro liters reactions containing 10 ng of the DNA template, 200 µM of each dNTP, 200 nM of each primer, 2.5 mM of MgCl2, 1 U of GoTaq DNA polymerase, and 4 µl of the supplied PCR buffer. Equal amounts of the purified amplicons were combined to create three sequencing pools (20 samples per pool). Each sequencing pool was sequenced in a 1/16th region of a 454 reaction using a GS FLX sequencer at the University of Florida’s Interdisciplinary Center for Biotechnology Research.