Abstract
BackgroundC-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.ResultsTo 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.ConclusionThe 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.
Highlights
C-type cytoplasmic male sterility (CMS-C) is one of the three major types of cytoplasmic male sterility (CMS) in maize
To measure the expression levels of the tricarboxylic acid (TCA) cycle-related genes and validate the accuracy of the transcriptome sequencing, 12 and 9 genes were respectively selected to analyse their expression via quantitative PCR (qPCR). 18S RNA was used as an internal control
Lines that were nearly isogenic at the Rf4 locus were developed and characterized via genetic analysis, molecular marker detection and whole-genome sequencing
Summary
C-type cytoplasmic male sterility (CMS-C) is one of the three major types of cytoplasmic male sterility (CMS) in maize. Male-sterile cytoplasm can be divided into three categories according to the fertility restoration patterns displayed in the F1 hybrids: Texas (T), USDA (S) and Charrua (C) [7] Among plants of these categories, C-type cytoplasmic male sterility (CMS-C) presents a stable male sterility and positively affects grain yield, contributing to its great application value in maize hybrid seed production [8, 9]. Previous studies have indicated that, with the exception of the two dominant Rf4 and Rf5 genes, quantitative trait loci (QTLs) are involved in maize CMS-C fertility restoration [10,11,12] These QTLs always partially restore CMS-C male fertility, which makes their use difficult. Additional experimental evidence is needed to confirm the Rf4 candidate gene and to elucidate its functional mechanism
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