ena-STUDY-UNIVERSIDAD DE JAEN-13-11-2018-09:45:30:956-144
Ectomycorrhizal communities of Abies pinsapo conifer trees
In the spring of 2016 we collected root tips from A.pinsapo trees following the trunk to the superficial secondary roots. We excavated carefully to do not break or lose the mycorrhized fine roots, which usually are inside soil aggregates.The samples were preserved and transported in cold conditions to the laboratory in the following 12 hours, and then they were kept at -80°C until further analysis.Prior to DNA extraction, root samples were ground for 15 minutes at 500 rpm with a Retsch PM 100 (Retsch GmbH, Haan, Germany). DNA was extracted from 0.25 g of homogenized roots using the PowerSoil® DNA Isolation Kit (MO BIO Laboratories, Inc., Carlsbad, USA) following manufacturer's instructions. DNA quantity and quality was measured using a NanoDrop Spectrophotometer (Thermo Scientific NanoDrop 2000c). DNA samples were checked for amplification by means of the next PCR protocol: 1 µL of template DNA, 1 µL of each primer (10 µM), 5 µL of MyTaq™ Reaction Buffer (BIOLINE, London, UK), 0.125 µL of MyTaq™ polymerase (BIOLINE, London, UK) for a final volume of 25 µL. Amplification was run on a Thermal Cycler (BIO-RAD Peltier Thermal Cycler, London, UK) under the following conditions: 95ºC for 3 min, 34 cycles of 15 s at 95ºC, 55ºC for 15 s and 72ºC for 15 s.     DNA samples were submitted to the Ramacciotti Centre for Genomics (The University of New South Wales, Sydney, Australia) for PCR amplification and high throughput sequencing using the Miseq System (Illumina Inc, San Diego, CA, USA). The fungal ITS2 region was amplified using primers fITS7 and ITS4 (Ihrmark et al 2012). An equimolar pool of amplicons was purified twice with an AMPure XP kit (Agencourt, Takeley, United Kingdom) and sequenced using the MiSeq Reagent kit v2 (Illumina) in one 2 x 250 bp paired-end reads run (that included a total of 303 samples, 70 from this study and 233 from another study).
In the spring of 2016 we collected root tips from A.pinsapo trees following the trunk to the superficial secondary roots. We excavated carefully to do not break or lose the mycorrhized fine roots, which usually are inside soil aggregates.The samples were preserved and transported in cold conditions to the laboratory in the following 12 hours, and then they were kept at -80°C until further analysis.Prior to DNA extraction, root samples were ground for 15 minutes at 500 rpm with a Retsch PM 100 (Retsch GmbH, Haan, Germany). DNA was extracted from 0.25 g of homogenized roots using the PowerSoil® DNA Isolation Kit (MO BIO Laboratories, Inc., Carlsbad, USA) following manufacturer's instructions. DNA quantity and quality was measured using a NanoDrop Spectrophotometer (Thermo Scientific NanoDrop 2000c). DNA samples were checked for amplification by means of the next PCR protocol: 1 µL of template DNA, 1 µL of each primer (10 µM), 5 µL of MyTaq™ Reaction Buffer (BIOLINE, London, UK), 0.125 µL of MyTaq™ polymerase (BIOLINE, London, UK) for a final volume of 25 µL. Amplification was run on a Thermal Cycler (BIO-RAD Peltier Thermal Cycler, London, UK) under the following conditions: 95ºC for 3 min, 34 cycles of 15 s at 95ºC, 55ºC for 15 s and 72ºC for 15 s.     DNA samples were submitted to the Ramacciotti Centre for Genomics (The University of New South Wales, Sydney, Australia) for PCR amplification and high throughput sequencing using the Miseq System (Illumina Inc, San Diego, CA, USA). The fungal ITS2 region was amplified using primers fITS7 and ITS4 (Ihrmark et al 2012). An equimolar pool of amplicons was purified twice with an AMPure XP kit (Agencourt, Takeley, United Kingdom) and sequenced using the MiSeq Reagent kit v2 (Illumina) in one 2 x 250 bp paired-end reads run (that included a total of 303 samples, 70 from this study and 233 from another study).