10 likes | 89 Views
DNA was extracted from forest soil, amplified with ITS primers and cloned. Sporocarps were collected and DNA was extracted. DNA was extracted from sub-sampled ECM root tips. Soil cores collected in June and September 2006 and all woody roots separated from soil cores.
E N D
DNA was extracted from forest soil, amplified with ITS primers and cloned. Sporocarps were collected and DNA was extracted. DNA was extracted from sub-sampled ECM root tips Soil cores collected in June and September 2006 and all woody roots separated from soil cores. All viable roots from each core were bead beaten to extract DNA, amplified with labeled ITS2 primers 58A2F (6FAM) and NLB4 (HEX). These 3 sources of environmental DNA were used for database construction. DNA was sequenced using ITS primers and fungi were identified by comparison to EMBL/GenBank/DDBJ database entries. PCR product was cut with restriction enzyme AluI and HaeIII to generate 4 TRFLP profiles per core. Since the NLB4 labeled primer generated the same size peak for most samples with AluI, we had 3 community profiles to use for identification of root fungi (below). DNA was used for PCR with labeled ITS2 primers. TRFLP with AluI and HaeIII resulted in 3 distinct TRFs that together comprise the “fingerprint” for the fungal type. Fragsort was used to identify fungi in complex communities using our database. AluI 58A2F 58A2F (AluI) 58A2F (HaeIII) NLB4 (HaeIII) Table S1 HaeIII 58A2F HaeIII NLB4