3730xl instrument

Ninety-six pairs of SSR primers with three bases and above were designed and synthesized by Tianyi Huiyuan Biotech Company, Wuhan, China (Table S1). The PCR reaction system contained 5.0 μL 2 × Taq PCR Master Mix, 1 μL template DNA (20 ng/μL), 0.5 μL of each primer (10 μL/mol), and DNase-/RNase-free deionized water 3.0 μL. Two-stage amplification programs were used. In the first stage, the pre-denaturation at 95 °C for 5 min caused the annealing temperature to gradually decrease from 62 °C to 52 °C, with a total of 10 cycles. The second stage included 25 amplification cycles, and the annealing temperature was 52 °C. In these two stages, the denaturation and extension steps remained unchanged for 30 s at 95 °C and 72 °C, respectively. After the second stage, the final extension was carried out at 72 °C for 20 min. Ninety-six pairs of primers were selected for PCR amplification and detected by 1% agarose gel electrophoresis. Finally, 13 SSR markers were obtained (Table 2). The PCR products were subjected to SSR analysis on an ABI 3730xl instrument, and then the genotype data were read using GeneMarker (Applied Biosystems).

Frozen polyps of B. europaea (N = 4 corals per Site) were powdered with a mortar pestle using liquid nitrogen^{79 (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 (psbA^ncr) from the chloroplast mini-circle genes of dinoflagellates^{80 (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 psbA^ncr for most Symbiodiniaceae^{81 (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 fragment^{82 (link)}. Direct Sanger sequencing on PCR amplified DNA was performed using Big Dye 3.1 reagents (Life Sciences) and the Applied Biosystems 3730XL instrument.

Both pair-end and mate-pair (3-kb insertion) libraries were constructed and sequenced using a HiSeq 2000 platform (Illumina, USA) in the Chinese National Human Genome Center, Shanghai. The acquired 2×100 bp reads were assembled by velvet software [10] (link). Identification of contigs representing parts of SCC elements was performed using BLAST tools [11] (link), using mapping marker genes such as orfX, mecA and the ccr gene complex. Gap closure was then performed using standard PCR and PCR product sequencing using a 3730XL instrument (Applied Biosystems, USA).