Abstract
Seagrass meadows are threatened by coastal development and global change. In the face of these pressures, molecular techniques such as reverse transcription quantitative real-time PCR (RT-qPCR) have great potential to improve management of these ecosystems by allowing early detection of chronic stress. In RT-qPCR, the expression levels of target genes are estimated on the basis of reference genes, in order to control for RNA variations. Although determination of suitable reference genes is critical for RT-qPCR studies, reports on the evaluation of reference genes are still absent for the major Australian species Zostera muelleri subsp. capricorni (Z. muelleri). Here, we used three different software (geNorm, NormFinder and Bestkeeper) to evaluate ten widely used reference genes according to their expression stability in Z. muelleri exposed to light limitation. We then combined results from different software and used a consensus rank of four best reference genes to validate regulation in Photosystem I reaction center subunit IV B and Heat Stress Transcription factor A- gene expression in Z. muelleri under light limitation. This study provides the first comprehensive list of reference genes in Z. muelleri and demonstrates RT-qPCR as an effective tool to identify early responses to light limitation in seagrass.
Highlights
Worldwide, seagrass meadows are declining at an estimated rate of > 7% p.a.2 due to a range of factors including eutrophication and degrading water quality from coastal development[11,12]
The real-time quantitative polymerase chain reaction (RT-qPCR) method is a technique of choice to quantify the expression of stress-inducible genes, being established in various organisms ranging from microorganisms such as bacteria or microalgae to fungi, plants and animals[34,35], from the single cell level[36] to tissue specific or on whole organism RNA extracts
In order to obtain reliable gene expression level data via RT-qPCR, it is critical to normalize the levels of target genes on the basis of reference genes that are known to be continuously expressed at the same levels, to control for the amount and quality of starting material, enzymatic efficiencies, and overall transcriptional activity
Summary
Seagrass meadows are declining at an estimated rate of > 7% p.a.2 due to a range of factors including eutrophication and degrading water quality from coastal development[11,12]. A molecular toolkit to identify light limitation in seagrass could be a valuable tool to quantify the prevailing light conditions and (i) to identify if plants are under light limitation before a reduction in density takes place and (ii) to determine how vulnerable a meadow would be to increased/prolonged light attenuation e.g. through stress associated with a dredging plume. Having such a tool could greatly benefit future seagrass management and conservation decisions[33]. The aims of the present study were (i) to identify and evaluate a set of reference genes and (ii) to provide a validated normalization strategy for early detection of light limitation in Z. muelleri growing in Gladstone Harbour (Queensland, Australia) using RT-qPCR
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