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.

The viviparous-1 (Vp1) gene from maize encodes a transcription factor involved in abscisic acid (ABA) signaling that is associated with seed dormancy and preharvest sprouting (PHS). Mis-splicing of wheat homologous Vp1 transcripts has been considered the main factor causing PHS sensitivity in wheat. A maize Vp1 gene including its promoter and coding sequence was used for Agrobacterium tumefaciens mediated transformation of an elite wheat cultivar, Zheng9023. Plants expressing the Vp1 gene displayed a genetically stable, significantly enhanced seed dormancy and PHS tolerance. A significant reduction of α-amylase activity of mature grains was detected in all the transgenic wheat plants. Furthermore, quantitative real-time polymerase chain reactions revealed that more transcripts for the genes involved in VP1/ABA signaling, such as the genes coding for wheat ABA-insensitivity, malate oxidoreductase, peroxiredoxin, and late embryogenesis abundant proteins, were concomitantly accumulated during seed development in the transgenic wheat plants compared with the non-transgenic Zheng9023. These results indicated a highly functional compensation of the Vp1 gene in wheat by an alien homolog from maize, providing a promising approach to breed wheat cultivars with improved tolerance for PHS through a genetic engineering process.Highlights► Heterologous expression of a maize Vp1 gene regulated by its own promoter in wheat. ► Enhancement of seed dormancy and PHS tolerance in wheat by genetic engineering. ► Seed dormancy and PHS tolerance closely correlated with α-amylase activity and expression of ABA signaling genes in transgenic wheat. ► Functional compensation of wheat Vp1 by a maize homolog. ► Generation of transgenic wheat using Pmi gene as selection marker mediated by Agrobacterium.