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

Abstract

Cytoplasmic male sterility (CMS) is a maternally inherited trait that results in the production of dysfunctional 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 stages 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 mitochondria F1F0-ATPase 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.

Identification of Appropriate Reference Genes for Normalizing miRNA Expression in Citrus Infected by Xanthomonas citri subsp. citri

“…Wu et al (2014) suggested that GAPDH was the most reliable reference gene in navel orange (C. sinensis L. Osbeck) fruit during five different ripening stages [25]. In contrast, our results indicated that GAPDH was unstable, and was not an ideal reference gene for gene expression analyses, which was consistent with the reports of the others [112][113][114][115][116], that GAPDH was an unstably expressed gene in different varieties of oranges under different conditions. In another study, a total of 22 GAPDH genes were identified in wheat.…”

Comparative Transcriptome Analysis Reveals the Cause for Accumulation of Reactive Oxygen Species During Pollen Abortion in Cytoplasmic Male-Sterile Kenaf Line 722HA

“…F 1 F 0 -ATPase (Complex V) is an important component of mitochondria and is reversibly involved in the synthesis and hydrolysis of ATP, depending on the direction of an electrochemical gradient that is formed by the passage of protons [34,35]. Many studies have shown that mitochondria encoded subunits of the F 1 F 0 -ATPase are associated with male sterility in plants [36,37].…”

Comparative Transcriptome Analysis between a Novel Allohexaploid Cotton Progeny CMS Line LD6A and Its Maintainer Line LD6B

“…In China, cotton production has been improved by artificial emasculation and pollination, however this required a long time, intensive labor and cost to achieve [2]. Cytoplasmic male sterility (CMS) is a classical and convenient way to improve crop yields in cotton [3][4][5], kenaf [6], sweet orange (Citrus sinensis) [7], rape (Brassica napus) [8] and chili pepper (Capsicum annuum L.) [9]. Producing superior varieties using the hybridization method improves crop yields, however, interspecific hybrid incompatibility occurs in distant hybridization.…”