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Microsporogenesis is an indispensable period for investigating microspore development and cytoplasmic male sterility (CMS) occurrence. Radish CMS line plays a critical role in elite F hybrid seed production and heterosis utilization. However, the molecular mechanisms of microspore development and CMS occurrence have not been thoroughly uncovered in radish. In this study, a comparative analysis of radish floral buds from a CMS line (NAU-WA) and its maintainer (NAU-WB) was conducted using next generation sequencing (NGS) technology. Digital gene expression (DGE) profiling revealed that 3504 genes were significantly differentially expressed between NAU-WA and NAU-WB library, among which 1910 were upregulated and 1594 were downregulated. Gene ontology (GO) analysis showed that these differentially expressed genes (DEGs) were mainly enriched in extracellular region, catalytic activity, and response to stimulus. KEGG enrichment analysis revealed that the DEGs were predominantly associated with flavonoid biosynthesis, glycolysis, and biosynthesis of secondary metabolites. Real-time quantitative PCR analysis showed that the expression profiles of 13 randomly selected DEGs were in high agreement with results from Illumina sequencing. Several candidate genes encoding ATP synthase, auxin response factor (ARF), transcription factors (TFs), chalcone synthase (CHS), and male sterility (MS) were responsible for microsporogenesis. Furthermore, a schematic diagram for functional interaction of DEGs from NAU-WA vs. NAU-WB library in radish plants was proposed. These results could provide new information on the dissection of the molecular mechanisms underlying microspore development and CMS occurrence in radish. [ABSTRACT FROM AUTHOR]

Cytoplasmic male sterility (CMS) is a maternally inherited trait, which becomes an important way in heterosis breeding to simplify the seed production procedures and reduce the production cost. Cytological observation of pollen development stages showed a clear difference between the newly developed CMS line and its maintainer; the profiling comparisons between floral buds of CMS line and its maintainer were conducted using differential display reverse transcription PCR (DDRT-PCR) technology. Thirty genes were up-regulated and sixty genes were down-regulated in newly developed CMS line when compared with its maintainer. These genes were involved in cell wall biosynthesis and regulation, transporter and ion channel, flower development and protein metabolism, etc. Expression patterns of six genes encoding RsCAM6, RsGPI, RsPMEI, RsRac, RsCHS, and RsSTP9 were verified by RT-PCR and qRT-PCR in different development stage of floral buds and different organs of CMS line and its maintainer. The expression level of RsRac was higher in stamens and microspores of CMS line than in maintainer. In comparison, the expression levels of the other five genes in CMS line were lower compared with maintainer. This expression profile suggests that these genes played important roles in the development of the pollen and may be closely related to male sterility. The results observed in this study will contribute to understanding the mechanism of pollen abortion during CMS in radish. [ABSTRACT FROM AUTHOR]

Cytoplasmic male sterility (CMS) is a maternally inherited trait that is widely utilized in hybrid seed production. In this study, the Rs- Rf gene and its allele, the Rs- rf gene, were isolated from a radish CMS restorer line and maintainer, respectively. The Rs- Rf gene was identified as a member of the pentatricopeptide repeat family. The results of reverse transcription (RT)-PCR and quantitative RT-PCR analyses of the floral buds at different development stages were used to characterize the expression features of Rs- Rf and orf138, a unique transcribed gene in the mitochondrion for its role in determining male sterility in the cytoplasm. orf138 was up-regulated in the CMS line and F hybrid at three bud developmental stages, including meiosis and the tetroid and microspore stages, but it was not expressed in the maintainer and restorer line, suggesting that the Rs- Rf gene was not able to block orf138 transcription. As in the F plant, the transcription of Rs- Rf was gradually up-regulated in the restorer line with progressive stages of bud development, but it was not transcribed in the CMS line and maintainer. Different restriction sites for SspI between the allele of Rs- Rf and Rs- rf were also identified, and a guideline for using the functional marker to distinguish the genotypes of individuals with Rs- Rf was developed. Due to its co-dominant character, this marker could be used to discriminate the heterozygous and homozygous fertility-restoring allele. Using this functional marker, we established a system of molecular marker-assisted development of elite CMS lines in radish. This system will contribute greatly to candidate maintainer identification and accelerate the process of elite CMS line development in late-bolting radish breeding programs. [ABSTRACT FROM AUTHOR]