Computational protocol: Who Is in There? Exploration of Endophytic Bacteria within the Siphonous Green Seaweed Bryopsis (Bryopsidales, Chlorophyta)

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[…] To determine the bacterial diversity, purified 16S rRNA gene amplicons from the algal extracts were cloned using the pGEM®-T Vector System (Promega Benelux, The Netherlands). For each Bryopsis sample a clone library of 150 clones was prepared, the diversity of which was examined via short fragment sequencing (see below). For dereplication, clones' short sequences were grouped into the same operational taxonomic unit (OTU) when having ≥97% similarity. From each OTU, representative clones were selected for full length (±1450 bp) 16S rRNA gene sequencing (see below). Clone libraries' coverage was verified by DGGE analysis of each Bryopsis DNA extract and its representative clones. A V3 PCR with primers F357-GC/R518 and subsequent DGGE analysis were carried out as described previously , with a denaturing gradient of 45–65%. DGGE banding patterns were normalized using the BioNumerics 5.1 software (Applied Maths, Belgium). DGGE bands from the algal extracts which showed no correspondence with OTU band positions were excised from the polyacrylamide gel following Van Hoorde et al. and sequenced (±150 bp) as described below. [...] Two sets of alignments, made using MUSCLE , were considered for phylogenetic analyses. The first one, consisting of a concatenated chloroplast 16S rRNA gene and rbcL dataset, was used for the creation of a Bryopsis phylogram. A second set of alignments was assembled to assess 16S rRNA gene phylogenetic relationships between the Bryopsis-associated bacterial endophytes and known bacterial species, including BLAST hits and algae-associated bacteria described in the literature. The most suitable model for phylogenetic analysis was selected using the AIC criterion in jModelTest . Subsequently, the Bryopsis host and bacterial datasets were analyzed by means of the maximum likelihood (ML) algorithm in PhyML v3.0 under a HKY + G4 model via the University of Oslo Bioportal website (http://www.bioportal.uio.no//). Reliability of ML trees was evaluated based on 100 bootstrap replicates. Output ML trees were subsequently visualized in Mega 4.0 and edited with Adobe® Illustrator® CS5. […]

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