Internal reference genes that are stably expressed are essential for normalization in comparative expression analyses. However, gene expression varies significantly among species, organisms, tissues, developmental stages, stresses, and treatments. Therefore, identification of stably expressed reference genes in developmental endosperm of bread wheat is important for expression analysis of endosperm genes. As the first study to systematically screen for reference genes across different developmental stages of wheat endosperm, nine genes were selected from among 76 relatively stable genes based on high-throughput RNA sequencing data. The expression stability of these candidate genes and five traditional reference genes was assessed by real-time quantitative PCR combined with three independent algorithms: geNorm, NormFinder, and BestKeeper. The results showed that ATG8d was the most stable gene during wheat endosperm development, followed by Ta54227, while the housekeeping gene GAPDH, commonly used as an internal reference, was the least stable. ATG8d and Ta54227 together formed the optimal combination of reference genes. Comparative expression analysis of glutenin genes indicated that credible quantification could be achieved by normalization against ATG8d in developmental endosperm. The stably expressed gene characterized here can act as a proper internal reference for expression analysis of wheat endosperm genes, especially nutrient- and nutrient synthesis–related genes.

中文翻译：

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内部参考基因的全基因组鉴定，用于小麦胚乳发育过程中基因表达值的标准化。

稳定表达的内部参考基因对于比较表达分析中的标准化至关重要。但是，基因表达在物种，生物体，组织，发育阶段，压力和治疗方法之间差异很大。因此，鉴定面包小麦发育胚乳中稳定表达的参考基因对于胚乳基因的表达分析很重要。作为首次系统筛选小麦胚乳不同发育阶段的参考基因的研究，基于高通量RNA测序数据，从76个相对稳定的基因中选择了9个基因。通过实时定量PCR结合三种独立的算法（geNorm，NormFinder和BestKeeper）评估了这些候选基因和五个传统参考基因的表达稳定性。在小麦胚乳发育过程中，ATG8d是最稳定的基因，其次是Ta54227，而通常用作内部参考的管家基因GAPDH却最不稳定。ATG8d和Ta54227一起形成了参考基因的最佳组合。谷蛋白基因的比较表达分析表明，通过标准化发育胚乳中的ATG8d可以实现可靠的定量。这里表征的稳定表达的基因可以作为小麦胚乳基因，特别是与营养素和营养素合成相关的基因的表达分析的适当内部参考。