Characterization of reference genes for RT-qPCR in the desert moss Syntrichia caninervis in response to abiotic stress and desiccation/rehydration.

Xiaoshuang Li,Haiyan Li,Bei Gao,Yuanming Zhang,Andrew J Wood,Daoyuan Zhang,Honglan Yang

doi:10.3389/fpls.2015.00038

Abstract

Syntrichia caninervis is the dominant bryophyte of the biological soil crusts found in the Gurbantunggut desert. The extreme desert environment is characterized by prolonged drought, temperature extremes, high radiation and frequent cycles of hydration and dehydration. S. caninervis is an ideal organism for the identification and characterization of genes related to abiotic stress tolerance. Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) expression analysis is a powerful analytical technique that requires the use of stable reference genes. Using available S. caninervis transcriptome data, we selected 15 candidate reference genes and analyzed their relative expression stabilities in S. caninervis gametophores exposed to a range of abiotic stresses or a hydration-desiccation-rehydration cycle. The programs geNorm, NormFinder, and RefFinder were used to assess and rank the expression stability of the 15 candidate genes. The stability ranking results of reference genes under each specific experimental condition showed high consistency using different algorithms. For abiotic stress treatments, the combination of two genes (α-TUB2 and CDPK) were sufficient for accurate normalization. For the hydration-desiccation-rehydration process, the combination of two genes (α-TUB1 and CDPK) were sufficient for accurate normalization. 18S was among the least stable genes in all of the experimental sets and was unsuitable as reference gene in S. caninervis. This is the first systematic investigation and comparison of reference gene selection for RT-qPCR work in S. caninervis. This research will facilitate gene expression studies in S. caninervis, related moss species from the Syntrichia complex and other mosses.

Highlights

Syntrichia caninervis is a desert moss and the dominant bryophyte of the biological soil crusts found in the Gurbantunggut desert of Northwestern China (Zhang, 2005)
For the hydration-desiccation-rehydration process, dry gametophores were fully hydrated by placing upon MINIQ-filtered water saturated filter paper (8 mL) in glass petri dishes for 24 h at 25◦C (150 μm m−2 s−1; Zhang et al, 2011b), transfer to clean dishes and dried at room temperature for 6 h (∼25◦C, RH = 25%; Yang et al, 2012), rehydrated by transferring to new petri plates and the filter paper was saturated with 8 mL filtered water at 25◦C for 2 and 6 h
SELECTION OF CANDIDATE REFERENCE GENES AND GENE SEQUENCE ANALYSIS The cDNA fragments of the 15 reference genes ranged from 480 bp for SAND to 2390 bp for calmodulin-like domain protein kinase (CDPK)

Summary

Introduction

Syntrichia caninervis is a desert moss and the dominant bryophyte of the biological soil crusts found in the Gurbantunggut desert of Northwestern China (Zhang, 2005). S. caninervis has gained particular attention due to its extreme desiccation tolerance (DT; Wood, 2007; Yang et al, 2012) and is closely related to Tortula ruralis. T. ruralis is a model DT moss and many desiccation-related genes have been isolated and analyzed in this species T. ruralis (Chen et al, 2002; Chen and Wood, 2003; Peng et al, 2005). EST data from desiccated and rehydrated T. ruralis gametophytes indicated many novel genes exist in this DT species (Wood et al, 1999; Oliver et al, 2004). S. caninervis, as compared to T. ruralis, is reported to be more tolerant to desiccation stress and have a quicker recovery rate from complete water loss (Oliver et al, 1993; Zhang et al, 2011b). S. caninervis is tolerant to multiple stresses including drought, high/low temperature, high radiation and frequent cycles of hydration and dehydration (Yang et al, 2012)

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Results

Conclusion