Genome-Wide Identification and Evaluation of New Reference Genes in Pineapple ( Ananas comosus L .) during Stamen and Ovule Development

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.