Selection and Validation of Appropriate Reference Genes for Quantitative Real-Time PCR Normalization in Staminate and Perfect Flowers of Andromonoecious Taihangia rupestris

作者：Weiguo Li, Lihui Zhang, Yandi Zhang, Yandi Zhang, Guodong Wang, Dangyu Song, Yanwen Zhang, Yanwen Zhang · 发表于：Frontiers in Plant Science · 年份：2017 · DOI：10.3389/fpls.2017.00729 · 被引用次数：36 · 研究领域：Molecular Biology Techniques and Applications、Environmental DNA in Biodiversity Studies、RNA Research and Splicing

Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is the most commonly used and powerful method for gene expression analysis due to its high sensitivity, specificity, and high throughput, and the accuracy of this approach depends on the stability of reference genes used for normalization. Taihangia rupestris Yu & Li (Rosaceae), an andromonoecious plant, produces both bisexual flowers and unisexual male flowers within the same individual. Using qRT-PCR technique, investigation of the gene expression profiling in staminate and perfect flowers would improve our understanding of the molecular mechanism in regulation of flower formation and sex differentiation in andromonoecious T. rupestris. To accurate normalize the gene expression level in Taihangia flower, 16 candidate reference genes, including 10 traditional housekeeping genes and 6 newly stable genes, were selected based on transcriptome sequence data and previous studies. The expressions of these genes were assessed by qRT-PCR analysis in 51 floral samples, including floral tissues in mature flowers, staminate and perfect flowers across developmental stages. By using geNorm, NormFinder, BestKeeper, and comprehensive RefFinder algorithms, ADF3 combined with UFD1 were identified as the optimal reference genes for staminate flowers, while the combination of HIS3/ADF3 was the most accurate reference genes for perfect and all floral samples, respectively. For floral tissues, HIS3/UFD1was the most ...