Evaluation of Four Endogenous Reference Genes and Their Real-Time PCR Assays for Common Wheat Quantification in GMOs Detection

<div><p>Proper selection of endogenous reference genes and their real-time PCR assays is quite important in genetically modified organisms (GMOs) detection. To find a suitable endogenous reference gene and its real-time PCR assay for common wheat (<i>Triticum aestivum</i> L.) DNA content or copy number quantification, four previously reported wheat endogenous reference genes and their real-time PCR assays were comprehensively evaluated for the target gene sequence variation and their real-time PCR performance among 37 common wheat lines. Three SNPs were observed in the <i>PKABA1</i> and <i>ALMT1</i> genes, and these SNPs significantly decreased the efficiency of real-time PCR amplification. GeNorm analysis of the real-time PCR performance of each gene among common wheat lines showed that the <i>Waxy-D1</i> assay had the lowest M values with the best stability among all tested lines. All results indicated that the <i>Waxy-D1</i> gene and its real-time PCR assay were most suitable to be used as an endogenous reference gene for common wheat DNA content quantification. The validated <i>Waxy-D1</i> gene assay will be useful in establishing accurate and creditable qualitative and quantitative PCR analysis of GM wheat.</p>