25-SEYITS2
Triplicate fragments were sampled for each of four key coral species found throughout the upper reef slope at both turbid and clear water sites; A. gemmifera, A. muricata, P. lutea and C. aspera (3 fragments x 4 species x 2 sites x 2 coral condition = 48 fragments total). Each replicate sample was taken from independent colonies. Small subsamples were taken from each fragment for Symbiodinium cell density assessment using a haemocytometer and normalized to surface area. Fragments were then preserved in RNAlater for symbiont ITS2 genotyping. Coral fragments were thawed slowly on ice and removed from the RNAlater solution using sterile forceps and kimwipes to remove excess solution. Coral fragments were transferred into sterile zip-lock bags and subsequently air-blasted using airflow from a sterile pipette tip (1000 L filter barrier tips, Neptune, USA) into 5 ml PBS-EDTA with care was taken to exclude any skeletal fragments. DNA was extracted from the tissue slurry using the Qiagen DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) according to manufacturer's instructions. To disrupt Symbiodinium cells, 200 l tissue slurry in 200 l AP-1, 4 l RNase A stock solution (100 g/ml), and 200 l 0.5 mm sterile glass beads (BioSpec, Bartlesville, OK) were bead-beaten at 30 Hz for 90 s with a Tissue Lyser II (Qiagen, Hilden, Germany). An additional step using phenol:chloroform:isoamyl alcohol (25:24:1) pre-buffered to pH 8 was used to purify the DNA. Extracted DNA was quantified and quality checked using a NanoDrop 2000C spectrophotometer (Thermo Fisher Scientific, Waltham, MA). PCR amplifications were performed in triplicate reactions with Qiagen Multiplex PCR Kit (Qiagen, Hilden, Germany). For Symbiodinium typing, we used the primers ITSintfor2 5'- TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG GAA TTG CAG AAC TCC GTG-3' and ITS2-reverse 5'- GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA GGG GAT CCA TAT GCT TAA GTT CAG CGG GT-3' with concatenated Illumina sequencing adapters (underlined). Initial PCR amplification was achieved with the following thermal cycles: 94C for 15 min, then 35 cycles of 94C for 30 s, 51C for 30 s, 72C for 30 s, followed by one cycle of 72C for 10 min and 4C hold. Sample triplicates were subsequently pooled and then purified using the Agencourt AMPure XP magnetic bead system (Beckman Coulter, Brea, CA, USA). Purified PCR products were subjected to an indexing PCR (8 cycles) to add Nextera XT indexing and sequencing adapters (Illumina) according to the manufacturer's protocol. Indexed amplicons were again purified, quantified on the QuBit (Quant-IT dsDNA Broad Range Assay Kit; Invitrogen, Carlsbad, CA, USA) and pooled in equimolar ratios on the BioAnalyzer (Agilent Technologies, Santa Clara, CA, USA). The final pooled library was purified on a 2% agarose gel to remove excess primer dimer. The library was sequenced at 8pM with 10% phiX on the Illumina Miseq, 2x 300 bp end version 3 chemistry according to the manufacturer's specifications at the Bioscience Core lab (KAUST, Saudi Arabia).