Selection of Reference Genes for qRT-PCR Analysis of Gene Expression in <i>Stipa grandis</i> during Environmental Stresses

<div><p><i>Stipa grandis</i> P. Smirn. is a dominant plant species in the typical steppe of the Xilingole Plateau of Inner Mongolia. Selection of suitable reference genes for the quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) is important for gene expression analysis and research into the molecular mechanisms underlying the stress responses of <i>S</i>. <i>grandis</i>. In the present study, 15 candidate reference genes (<i>EF1 beta</i>, <i>ACT</i>, <i>GAPDH</i>, <i>SamDC</i>, <i>CUL4</i>, <i>CAP</i>, <i>SNF2</i>, <i>SKIP1</i>, <i>SKIP5</i>, <i>SKIP11</i>, <i>UBC2</i>, <i>UBC15</i>, <i>UBC17</i>, <i>UCH</i>, and <i>HERC2</i>) were evaluated for their stability as potential reference genes for qRT-PCR under different stresses. Four algorithms were used: GeNorm, NormFinder, BestKeeper, and RefFinder. The results showed that the most stable reference genes were different under different stress conditions: <i>EF1beta</i> and <i>UBC15</i> during drought and salt stresses; <i>ACT</i> and <i>GAPDH</i> under heat stress; <i>SKIP5</i> and <i>UBC17</i> under cold stress; <i>UBC15</i> and <i>HERC2</i> under high pH stress; <i>UBC2</i> and <i>UBC15</i> under wounding stress; <i>EF1beta</i> and <i>UBC17</i> under jasmonic acid treatment; <i>UBC15</i> and <i>CUL4</i> under abscisic acid treatment; and <i>HERC2</i> and <i>UBC17</i> under salicylic acid treatment. <i>EF1beta</i> and <i>HERC2</i> were the most suitable genes for the global analysis of all samples. Furthermore, six target genes, <i>SgPOD</i>, <i>SgPAL</i>, <i>SgLEA</i>, <i>SgLOX</i>, <i>SgHSP90</i> and <i>SgPR1</i>, were selected to validate the most and least stable reference genes under different treatments. Our results provide guidelines for reference gene selection for more accurate qRT-PCR quantification and will promote studies of gene expression in <i>S</i>. <i>grandis</i> subjected to environmental stress.</p>