Wheat-Specific Gene, <i>Ribosomal Protein L21</i>, Used as the Endogenous Reference Gene for Qualitative and Real-Time Quantitative Polymerase Chain Reaction Detection of Transgenes

Wheat-specific <i>ribosomal protein L21</i> (<i>RPL21</i>) is an endogenous reference gene suitable for genetically modified (GM) wheat identification. This taxon-specific <i>RPL21</i> sequence displayed high homogeneity in different wheat varieties. Southern blots revealed 1 or 3 copies, and sequence analyses showed one amplicon in common wheat. Combined analyses with sequences from common wheat (AABBDD) and three diploid ancestral species, Triticum urartu (AA), Aegilops speltoides (BB), and Aegilops tauschii (DD), demonstrated the presence of this amplicon in the AA genome. Using conventional qualitative polymerase chain reaction (PCR), the limit of detection was 2 copies of wheat haploid genome per reaction. In the quantitative real-time PCR assay, limits of detection and quantification were about 2 and 8 haploid genome copies, respectively, the latter of which is 2.5–4-fold lower than other reported wheat endogenous reference genes. Construct-specific PCR assays were developed using <i>RPL21</i> as an endogenous reference gene, and as little as 0.5% of GM wheat contents containing Arabidopsis <i>NPR1</i> were properly quantified