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Background  C-type cytoplasmic male sterility (CMS-C) is one of the three major types of cytoplasmic male sterility (CMS) in maize.  Rf4  is a dominant restorer gene for CMS-C and has great value in hybrid maize breeding, but little information concerning its functional mechanism is known.    Results  To reveal the functional mechanism of  Rf4 , we developed a pair of maize near-isogenic lines (NILs) for the  Rf4  locus, which included a NIL_ rf4  male-sterile line and a NIL_ Rf4  male fertility-restored line. Genetic analysis and molecular marker detection indicated that the male fertility of NIL_ Rf4  was controlled by  Rf4 . Whole-genome sequencing demonstrated genomic differences between the two NILs was clustered in the  Rf4  mapping region. Unmapped reads of NILs were further assembled to uncover  Rf4  candidates. RNA-Seq was then performed for the developing anthers of the NILs to identify critical genes and pathways associated with fertility restoration. A total of 7125 differentially expressed genes (DEGs) were identified. These DEGs were significantly enriched in 242 Gene Ontology (GO) categories, wherein 100 DEGs were involved in pollen tube development, pollen tube growth, pollen development, and gametophyte development. Homology analysis revealed 198 male fertility-related DEGs, and pathway enrichment analysis revealed that 58 DEGs were enriched in cell energy metabolism processes involved in glycolysis, the pentose phosphate pathway, and pyruvate metabolism. By querying the Plant Reactome Pathway database, we found that 14 of the DEGs were involved in the mitochondrial tricarboxylic acid (TCA) cycle and that most of them belonged to the isocitrate dehydrogenase (IDH) and oxoglutarate dehydrogenase (OGDH) enzyme complexes. Transcriptome sequencing and real-time quantitative PCR (qPCR) showed that all the above TCA cycle-related genes were up-regulated in NIL_ Rf4 . The results of our subsequent enzyme-linked immunosorbent assay (ELISA) experiments pointed out that the contents of both the IDH and OGDH enzymes accumulated more in the spikelets of NIL _Rf4  than in those of NIL_ rf4.Conclusion  The present research provides valuable genomic resources for deep insight into the molecular mechanism underlying CMS-C male fertility restoration. Importantly, our results indicated that genes involved in energy metabolism, especially some mitochondrial TCA cycle-related genes, were associated with maize CMS-C male fertility restoration.    Electronic supplementary material  The online version of this article (10.1186/s12870-018-1409-z) contains supplementary material, which is available to authorized users.

Comparative transcriptome analysis reveals that tricarboxylic acid cycle-related genes are associated with maize CMS-C fertility restoration