Powerplant pro dna isolation kit

DNA was extracted from excised embryos of five germinated seeds of WT Bowman, WT Steptoe, nec3- γ1 (Bowman background), nec3.l (Steptoe background), and nec3.m (Steptoe background) mutants. DNA extractions were performed on mechanically lysed samples using the PowerPlant Pro DNA isolation kit (Qiagen, CA), following the protocol described by Solanki et al. [57 (link)].

An SDS-based method (modified from Tai and Tanksley [20 (link)]) and a CTAB-based method (modified from Chee et al. [41 ]) conventionally used for sugarcane and citrus (3.0 g of tissue), respectively, were tested in parallel with the TENS-CO method (0.15 g of tissue) for DNA extraction. Two commercial kits, PowerPlant Pro DNA Isolation Kit (SDS-based; MO BIO Laboratories; for rice, potato and tomato) (0.15 g of tissue) and Synergy Plant DNA Extraction Kit (CTAB-based; OPS Diagnostics, Lebanon, NJ, USA; for potato and tomato) (0.05 g of tissue) were also tested.

DNA for the enriched and unenriched rhizosphere samples was extracted using the PowerSoil DNA isolation kit (Mo Bio Laboratories, Carlsbad, CA) using the provided protocol. DNA for the enriched and unenriched endosphere samples was extracted using the PowerPlant Pro DNA isolation kit with phenolic removal protocol (Mo Bio Laboratories, Carlsbad, CA) using the provided protocol.

Total endophytic community DNA was extracted from surface disinfected root samples using the PowerPlant^®Pro DNA Isolation Kit (MoBio Laboratories Inc., Carlsbad, CA, USA) and the rhizosphere soil community DNA was extracted using the MoBio PowerSoil^® extraction kit (MoBio Laboratories Inc., Carlsbad, CA, USA). DNA exactions were conducted following the manufacture’s protocols. The DNA yield was quantified using Qubit^®Fluorometric Quantitation (Invitrogen) and in a SYBR Safe (Invitrogen) 1% agarose gel by comparison with a high DNA mass ladder (Invitrogen) using a Bio-Rad Gel Doc XR System (Bio-Rad Laboratories, Mississauga, ON, Canada).

DNA was extracted from two duplicate 0.5 g crown tissue samples of each plant using the Power Plant Pro DNA isolation kit (MO BIO Laboratories Inc., Carlsbad, CA, USA). Strawberry crown infection was determined by qPCR for M. phaseolina using previously described methods [30 (link)]. F. oxysporum f. sp. fragariae infection was assessed by qPCR using the specific primer pair FofraF (5′-CAG ACT GGG GTG CTT AAA GTT-3′)/FofraR (5′-AAC CGC TAG GGT CGT AAC AAA-3′) [35 (link)]. Plants were also processed for pathogen detection and isolation from plant tissue using the protocol modified from [36 (link)]. Surface disinfested crown tissue was finely cross-sectioned with a sterile razor blade, and tissues were used for DNA extraction, and fungal isolation on 1/10th-strength PDA amended with rifampicin (100 μg mL⁻¹, GoldBio, St Louis, MO, USA) and kanamycin (75 µg mL⁻¹). Plates were incubated at room temperature, and crown tissue was examined daily for the emergence of hyphal growth using a light microscope (100×). F. oxysporum f. sp. fragariae infection was assessed by qPCR or the crown plating method. Morphological features including M. phaseolina sclerotia and F. oxysporum f. sp. fragariae conidia, developing from fungal growth emanating from crowns plated on culture media, were used to document the recovery of the pathogen.

Total DNA was extracted from 250 mg of bulk or rhizosphere soil with MoBio’s PowerSoil DNA Isolation Kit (MO BIO Laboratories, Inc, Carlsbad, CA). Three grams of root material were pulverized with the Geno Grinder (HORIBA Canada Inc., London, Ontario) and 50 mg were extracted using the PowerPlant Pro DNA Isolation Kit (MO BIO Laboratories, Inc.). DNA samples were quantified by fluorescence detection using the Life Technologies Qubit^® dsDNA HS quantitation kit (Invitrogen, Waltham, MA). Libraries for sequencing were prepared according to Illumina’s “16 S Metagenomic Sequencing Library Preparation” guide (Part#15044223Rev.B). Amplicon libraries for the bacterial 16 S rRNA gene were prepared using primers F343 and R803 [42 (link)] whereas for the fungal ITS1 region the primers ITS1F and 58A2R [43 (link)] were used. We also used the gene coding for the group I chaperonins (CPN60, also known as GroEL or hsp60) to have an independent confirmation of the 16 S rRNA gene results. The cpn60 UT was amplified using the type I chaperonin universal primer cocktail containing a 1:3 ratio of H279/H280:H1612/H1613 as previously described [44 (link), 45 ]. The three pools were then loaded on an Illumina MiSeq sequencer and sequenced in-house using a 600-cycles MiSeq Reagent Kit v3.