Quantitative real-time PCR (qRT-PCR) is a convenient tool for gene expression analysis. However, reliability of its result is influenced largely by the selection of reference genes. Pineapple molecular breeding is limited due to lack of understanding of sexual organs’ development. Similarly, only a few reference genes have identified for qRT-PCR analysis in pineapple stamen and ovule. In this study, we initially identified 20 candidate reference genes using transcriptome data, and determined their expression stabilities in 36 ovule and stamen developmental samples using qRT-PCR. Three algorithms, GeNorm, NormFinder, and BestKeeper were used to determine the superiority of those candidate genes. Our results revealed that the use of combined RPS4 and RPL23 during ovule development, CCR and RPS4 during stamen development were sufficient for reliable normalization. These recommended reference genes were further verified by evaluating the temporal expression abundance of the meiosis-specific protein-encoding genes AcASY1 and AcASY3 in all experimental samples. Our results complement previous pineapple normalization study by providing appropriate reference genes during the entire reproductive period, and will beneficial for future studies on the pineapple stamen and ovule development. Finally, this result will help for the molecular breeding of pineapple for crop improvement.

实时定量PCR（qRT-PCR）是用于基因表达分析的便捷工具。但是，其结果的可靠性在很大程度上受参考基因的选择影响。由于缺乏对性器官发育的了解，菠萝分子育种受到限制。同样，在菠萝雄蕊和胚珠中仅鉴定了少数参考基因用于qRT-PCR分析。在这项研究中，我们最初使用转录组数据鉴定了20个候选参考基因，并使用qRT-PCR确定了它们在36个胚珠和雄蕊发育样品中的表达稳定性。使用三种算法GeNorm，NormFinder和BestKeeper来确定那些候选基因的优越性。我们的结果表明，在胚珠发育过程中结合使用RPS4和RPL23，雄蕊发育期间的CCR和RPS4足以实现可靠的标准化。通过评估所有实验样品中减数分裂特异蛋白质编码基因AcASY1和AcASY3的时间表达丰度，进一步验证了这些推荐的参考基因。我们的研究结果通过在整个生育期提供适当的参考基因，对先前的菠萝标准化研究进行补充，并将有助于未来对菠萝雄蕊和胚珠发育的研究。最后，该结果将有助于菠萝分子育种以改善作物。