Computational protocol: Transcriptional Response of Two Core Photosystem Genes in Symbiodinium spp. Exposed to Thermal Stress

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[…] presents a list of the real-time PCR primers used in this study. The Proliferating Cell Nuclear Antigen (PCNA) primers were previously established for Symbiodinium , while S-adenosyl-L-methionine synthetase (SAM) primers were selected due to their high expression stability in other Symbiodinium types exposed to both temperature and light stress . Primers for psbA and β-Actin were designed based on sequence alignments of cDNA from multiple Symbiodinium types, as well as, several other dinoflagellates obtained from the NCBI GenBank database (www.ncbi.nlm.nih.gov). Multiple sequence alignments were performed in BioEdit using the ClustalW Multiple Alignment tool . Primer sets were designed from these alignments within conserved regions identified for each gene using DNASTAR (Lasergene Primer Select). Primer compatibility was tested against DNA isolated from distinct Symbiodinium cultures that span five different clades (ITS2-types: A13, A20, B1, C1b-c, D1 and F2). An additional psbA primer set was designed for the in hospite Symbiodinium analysis due to the amplification of a second product (also derived from the psbA gene; data not shown) in C1b-c samples. Furthermore, the β-Actin primers failed to amplify within Symbiodinium A20, while the PCNA primers only weakly amplified cDNA from the D1 alga.Given the absence of any psaA sequence data for Symbiodinium, the psaA amino-acid sequence from the following microalgae were obtained from GenBank to create a multiple-sequence protein alignment: Glaucocystis nostochinearum (AAX82908.1), Gonyaulax polyedra (ABB89032.1), Heterocapsa triquetra (AAD44698.1), Heterocapsa niei (AAX82907.1), Amphidinium operculatum (CAB75844.1), Gymnodinium mikimotio (BAA86297.1), Neoceratium horridum (CAF32304.1), and Akashiwo sanguinea (AAX82906.1). Degenerate primers were designed (Forward (5′-3): CCTTCGGCCTGTACATCCAYAAYGAYAC; Reverse (5′3′): TGCAGTGGATTGTGAAGGCRTGNAYRTG) from the resulting protein alignment with CODEHOP (consensus-degenerate hybrid oligonucleotide primers; http://bioinformatics.weizmann.ac.il/blocks/codehop.html), and used to successfully amplify a 366 bps product from a Symbiodinium B1 (Conditions: 92°C for 3 min; 35 cycles of 92°C for 45 s, 61°C for 45 s, and 72°C for 45 s). The PCR product was cloned with a TOPO® TA Cloning kit with One Shot® Chemically Competent cells (Invitrogen) and bi-directionally sequenced using a BigDye® Terminator v1.1 Cycle Sequencing kit (Applied Biosystems). A BLAST search (blastn) of GenBank returned over 50 known psaA sequences with E-values less than 10−15. These hits matched sequences from various photosynthetic organisms, including the species that constructed the original protein alignment. Real-time PCR primers were developed from the resulting psaA sequence using DNASTAR Lasergene Primer Select and successfully amplified across several Symbiodinium samples spanning different clades (A, B, C, D and F). [...] Real-time PCR assays were performed on an ABI Prism 7500 Sequence Detection System (Applied Biosystems). Three technical replicates of each cDNA sample were amplified using 2 µL H2O, 5 µL SensiMix SYBR mastermix (Bioline), 1 µL of each forward and reverse primer (optimized final concentrations of 0.9 µM and 0.3 µM, respectively, for all primers), and 1 µL cDNA in 10 µL total volume reactions. PCR conditions consisted of 50°C for 2 min, 95°C for 10 min, followed by 40 cycles of 95°C for 15 s, 53°C for 30 s (48°C for the psaA primers), and 72°C for 1 min. Following each reaction, a dissociation step (melt curve analysis) from 60°C to 95°C was included to ensure that primer dimers did not significantly contribute to the fluorescence signal. In addition to the experimental samples, a five-step ten-fold dilution series of cDNA from a common control sample was included on each plate for every primer set to account for differences in priming efficiency between plates. A fixed fluorescent threshold of 0.05 was applied for all reactions to determine the cycle threshold (CT) values. The resulting CT values of the technical replicates were averaged and used only if their standard deviation was less than 0.50 (typically less than 0.25). Additionally, the expression stability of the three reference genes under each set of experimental conditions was evaluated using the open source software geNorm (http://medgen.ugent.be/~jvdesomp/genorm/). All housekeeping genes received an Expression Stability value (M) of less than 1.5 and were thereby considered adequate reference genes for a particular experiment . All psbA and psaA values were normalized to the geometric mean of three reference genes (SAM, Actin and PCNA) and expressed relative to untreated (control) samples taken at the same time as the treatment. As previously mentioned, the β-Actin and PCNA primers failed to amplify Symbiodinium A20 and D1, respectively. Therefore, these Symbiodinium types were only normalized against two of the three housekeeping genes. [...] All relative changes in gene expression were analyzed using the relative expression software tool, REST 2009 (http://www.qiagen.com/products/rest2009software.aspx). This software employs a statistical randomization technique to test for up or downregulation of a gene, based on a mean expression value normalized against multiple reference genes, with 10,000 iterations. For each Symbiodinium culture, differences in Fv/Fm and σPSII from the untreated controls (Day 0) were tested using a one-way analysis of variance (ANOVA) in SPSS version 19.0. Data were tested for assumptions of normality (Sharpiro-Wilk) and homogeneity of variance (Levene's test), and when applicable, a Tukey's multiple comparison test analyzed the intraspecific differences of each fluorescence metric. For the in hospite Symbiodinium measurements, sample variance was not homogeneous at all time points (Levene's test; p<0.05), and therefore, differences in Fv/Fm and σPSII between treatment and control corals for a particular Symbiodinium type were determined using the nonparametric Mann-Whitney U test. […]

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