Candidate Reference Genes Selection and Application for RT-qPCR Analysis in Kenaf with Cytoplasmic Male Sterility Background

Cytoplasmic male sterility (CMS) is a maternally inherited trait that results in the production of disfunctional pollen. Based on reliable reference gene-normalized real-time quantitative PCR (RT-qPCR) data, examining gene expression profile can provide valuable information on the molecular mechanism of kenaf CMS. However, studies have not been conducted regarding selection of reference genes for normalizing RT-qPCR data in the CMS and maintainer lines of kenaf crop. Therefore, we studied 10 candidate reference genes (ACT3, ELF1A, G6PD, PEPKR1, TUB, TUA, CYP, GAPDH, H3 and 18S) to assess their expression stability at three stage of pollen development in CMS line 722A and maintainer line 722B of kenaf. Five computational statistical approaches (GeNorm, NormFinder, ΔCt, BestKeeper and RefFinder) were used to evaluate the expression stability levels of these genes. According to RefFinder and GeNorm, the combination of TUB, CYP and PEPKR1 was identified as an internal control for the accurate normalization across all sample set, which was further confirmed by validating the expression of HcPDIL5-2a. Furthermore, the combination of TUB, CYP and PEPKR1 was used to differentiate the expression pattern of five ATP synthase subunit genes (atp1, atp4, atp6, atp8 and atp9) by RT-qPCR during pollen development in CMS line 722A and maintainer line 722B. We found that atp1, atp6 and atp9 exhibited significantly different expression patterns during pollen development in line 722A compared with line 722B. This is the first systematic study of reference genes selection for CMS and will provide useful information for future research on the gene expressions and molecular mechanisms underlying CMS in kenaf.