Frozen polyps of B. europaea (N = 4 corals per Site) were powdered with a mortar pestle using liquid nitrogen79 (link). DNA was isolated with the Wizard Genomic DNA Purification kit (Promega) according to the manufacturer’s instructions. Quality and quantity of extracted DNA was double checked using electrophoresis (0.8% agarose gel) and spectrophotometric measurements (λ=260 nm/280 nm).
The high resolution psbA non-coding region (psbAncr) from the chloroplast mini-circle genes of dinoflagellates80 (link),81 (link) was amplified and then directly sequenced. The ‘universal’ primers psbAFor_1 (5´GCA GCT CAT GGT TAT TTT GGT AGA C 3´) and psbARev_1 (5´AAT TCC CAT TCT CTA CCC ATC C 3´), designed to amplify the psbAncr for most Symbiodiniaceae81 (link), were used with the following PCR conditions: 94 °C for 2 min; then 40 cycles of 94 °C 10 s, 55 °C for 30 s and 72 °C for 2 min; and a final extension at 72 °C for 10 min. The internal primers Philozoon-psbAF (5´ATT TGG TTC ACA GCG CTT GG 3´) and Philozoon-psbAR (5´CCA TTT GAC TCC CAC ACT GGA) were also used for nucleotide sequencing through the middle region of the amplified fragment82 (link). Direct Sanger sequencing on PCR amplified DNA was performed using Big Dye 3.1 reagents (Life Sciences) and the Applied Biosystems 3730XL instrument.
The high resolution psbA non-coding region (psbAncr) from the chloroplast mini-circle genes of dinoflagellates80 (link),81 (link) was amplified and then directly sequenced. The ‘universal’ primers psbAFor_1 (5´GCA GCT CAT GGT TAT TTT GGT AGA C 3´) and psbARev_1 (5´AAT TCC CAT TCT CTA CCC ATC C 3´), designed to amplify the psbAncr for most Symbiodiniaceae81 (link), were used with the following PCR conditions: 94 °C for 2 min; then 40 cycles of 94 °C 10 s, 55 °C for 30 s and 72 °C for 2 min; and a final extension at 72 °C for 10 min. The internal primers Philozoon-psbAF (5´ATT TGG TTC ACA GCG CTT GG 3´) and Philozoon-psbAR (5´CCA TTT GAC TCC CAC ACT GGA) were also used for nucleotide sequencing through the middle region of the amplified fragment82 (link). Direct Sanger sequencing on PCR amplified DNA was performed using Big Dye 3.1 reagents (Life Sciences) and the Applied Biosystems 3730XL instrument.
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