title: Wheat-specific gene, ribosomal protein l21, used as the endogenous reference gene for qualitative and real-time quantitative polymerase chain reaction detection of transgenes.
abstract: Wheat-specific ribosomal protein L21 (RPL21) is an endogenous reference gene suitable for genetically modified (GM) wheat identification. This taxon-specific RPL21 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 RPL21 as an endogenous reference gene, and as little as 0.5% of GM wheat contents containing Arabidopsis NPR1 were properly quantified.
authors.affiliation: Molecular Biotechnology Laboratory of Triticeae Crops, ‡College of Life Science and Technology, §College of Plant Science and Technology, and ∥National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University , Wuhan, Hubei 430070, People's Republic of China.
authors.forename: Yi-Ke
authors.initials: YK
authors.lastname: Liu
authors.forename: He-Ping
authors.initials: HP
authors.lastname: Li
authors.forename: Tao
authors.initials: T
authors.lastname: Huang
authors.forename: Wei
authors.initials: W
authors.lastname: Cheng
authors.forename: Chun-Sheng
authors.initials: CS
authors.lastname: Gao
authors.forename: Dong-Yun
authors.initials: DY
authors.lastname: Zuo
authors.forename: Zheng-Xi
authors.initials: ZX
authors.lastname: Zhao
authors.forename: Yu-Cai
authors.initials: YC
authors.lastname: Liao
mesh_terms: D018628:Gene Dosage; D010940:Plant Proteins / Q000235:genetics*; D030821:Plants, Genetically Modified / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012015:Reference Standards; D012269:Ribosomal Proteins; D013045:Species Specificity; D019076:Transgenes; D014908:Triticum / Q000235:genetics*
chemical_list: D010940:Plant Proteins; D012269:Ribosomal Proteins; C060232:ribosomal protein L21
keywords: Triticum aestivum; endogenous reference gene; genetically modified organisms; real-time PCR; ribosomal protein L21