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Circadian rhythms regulate many physiological, behavioral and reproductive processes. These rhythms are often controlled by light, and daily cycles of solar illumination entrain many clock regulated processes. In scleractinian corals a number of different processes and behaviors are associated with specific periods of solar illumination or non-illumination-for example, skeletal deposition, feeding and both brooding and broadcast spawning.We have undertaken an analysis of diurnal expression of the whole transcriptome and more focused studies on a number of candidate circadian genes in the coral Acropora millepora using deep RNA sequencing and quantitative PCR. Many examples of diurnal cycles of RNA abundance were identified, some of which are light responsive and damped quickly under constant darkness, for example, cryptochrome 1 and timeless, but others that continue to cycle in a robust manner when kept in constant darkness, for example, clock, cryptochrome 2, cycle and eyes absent, indicating that their transcription is regulated by an endogenous clock entrained to the light-dark cycle. Many other biological processes that varied between day and night were also identified by a clustering analysis of gene ontology annotations.Corals exhibit diurnal patterns of gene expression that may participate in the regulation of circadian biological processes. Rhythmic cycles of gene expression occur under constant darkness in both populations of coral larvae that lack zooxanthellae and in individual adult tissue containing zooxanthellae, indicating that transcription is under the control of a biological clock. In addition to genes potentially involved in regulating circadian processes, many other pathways were found to display diel cycles of transcription.
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???displayArticle.pmcLink???PMC3176305 ???displayArticle.link???PLoS One
Figure 1. Solexa sequencing data processing pipeline.Detailed steps involved in Solexa deep sequencing of coral larvae samples for both day and night (12 hour difference). Preparation of cDNA library, sequencing and generation of output were performed by the BC Genome Sciences Centre, while all other components were performed by authors.
Figure 2. QPCR analysis of candidate circadian gene expression in larvae.Larvae were exposed to either a 12∶12 LD treatment or 12∶12 DD treatment for 24 hours, and were analyzed with QPCR for rhythmicity in candidate circadian genes (a. clock, b. cryptochrome 1, c. cryptochrome 2, d. cycle, e. eyes absent, f. timeless). Relative fold changes (mean ± SEM of triplicate QPCR reactions) in RNA expression levels are presented based on QPCR analysis of the 2009 larval sample. Shaded areas represent periods of darkness, with the exception of DD samples, which were darkened for 24 hours. Bonferroni post-tests were performed to confirm statistical significant differences at each time point. * represents P<0.05, ** represents P<0.01, and *** represents P<0.001.
Figure 3. Comparison of larvae and adult diurnal gene expression. QPCR analysis of candidate circadian gene expression in larvae.Larvae and adult tissues were compared using QPCR, to determine similar patterns of gene expression over a 12∶12 LD treatment (a. clock, b. cryptochrome 1, c. cryptochrome 2, d. cycle, e. eyes absent, f. timeless). Relative fold changes in RNA expression levels (mean ± SEM of triplicate QPCR reactions) are presented for both 2009 larvae (primary y-axis) and adult tissue (secondary y-axis). Shaded areas represent the 12 hours of darkness for larvae (12∶12 LD) and 11 hours of darkness for adult tissue (13∶11 LD).
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