Glomus etunicatum spores PLS amplicon 454 sequencing
Two to three spores from each of three replicate host plants were isolated for further analysis. These spores were individually picked and placed in 1 µl sterile water in a 0.2 ml tube, then crushed using fine forceps, which were flame-sterilized between samples. The entire DNA content of each of the spores was amplified using the GenomiPhi Whole-Genome Amplification Kit (GE Healthcare, Canada) according to manufacturer instructions. PLS sequences were then amplified using DreamTaq DNA polymerase (Fermentas) using primers Pol4-A (GAATCCTTCCCAAATTGATCAGAATACTTGTT) and Pol7-B (TAATAATAAAAGCCTTTCAAAAAATCCATCAATA) [30], with added pyrosequencing adaptor and key sequences at the 5’ ends (forward: CCATCTCATCCCTGCGTGTCTCCGACTCAG, reverse: CCTATCCCCTGTGTGCCTTGGCAGTCTCAG). The reaction was performed in 50 µl volumes containing 0.2 mM dNTPs, 0.5 µmol of each primer and the PCR buffer. PCR was carried out for 34 cycles (95°C for 30 sec, 50°C for 30 sec, 72°C for 1 min; preceded by an initial 2 min denaturation at 94°C and followed by a 10 min hold at 72°C) on a Mastercycler ep gradient S thermal cycler (Eppendorf). The PCR product was checked on an electrophoresis gel to ensure successful amplification of the gene, then purified using a MinElute PCR Purification Kit (Qiagen) according to manufacturer instructions. These purified samples were then sent to the Genome Quebec Innovation Centre (McGill University, Montreal) for pyrosequencing using the GS FLX Titanium emPCR kit (Roche 454 Life Science) with lib-L chemistry (1/8 run per sample).