Twelve genes with differential expression from the 5 genomic regions identified on chromosome 2.

<p>Twelve genes with differential expression from the 5 genomic regions identified on chromosome 2.</p

Pollen staining with I₂-KI revealed the normal development of a small portion of pollen from the partially rescued FIL-B.

<p>Representative images of the stained pollen of Jing724 (<b>A</b>) and the partially rescued (<b>B</b>) and sterile (<b>C</b>) individuals of FIL-B. Pollen was collected when the anthers exserted in the partially rescued plants. Round pollen with black staining was recorded as normal. The scale bars represent 200 μm.</p

Identification of genomic regions contributing to the fertility instability in FIL-B by BSR-Seq using the Euclidean distance (ED) algorithm.

<p><b>A.</b> The ED scores raised to the fifth power across the genome. Each dot represents each SNP identified from the RNA-Seq, and the different colors designate the different chromosomes as indicated on the X-axis. For all of the panels, the gray vertical dotted lines delineate the chromosome edges, and the width of the chromosome represents the relative numbers of SNPs identified. The pink horizontal dotted lines represent the significant threshold of the 99% percentile of the ED⁵. <b>B.</b> The ED⁵ scores of a close-up of chromosome 2. <b>C.</b> The Loess fit curve calculated from A. <b>D.</b> The Loess fit curve of a close-up of chromosome 2 with the physical position indicated on X-axis. Each peak represents a possible associated genomic region.</p

Identification of Genes Potentially Associated with the Fertility Instability of S-Type Cytoplasmic Male Sterility in Maize via Bulked Segregant RNA-Seq

<div><p>S-type cytoplasmic male sterility (CMS-S) is the largest group among the three major types of CMS in maize. CMS-S exhibits fertility instability as a partial fertility restoration in a specific nuclear genetic background, which impedes its commercial application in hybrid breeding programs. The fertility instability phenomenon of CMS-S is controlled by several minor quantitative trait locus (QTLs), but not the major nuclear fertility restorer (<i>Rf3</i>). However, the gene mapping of these minor QTLs and the molecular mechanism of the genetic modifications are still unclear. Using completely sterile and partially rescued plants of fertility instable line (FIL)-B, we performed bulk segregant RNA-Seq and identified six potential associated genes in minor effect QTLs contributing to fertility instability. Analyses demonstrate that these potential associated genes may be involved in biological processes, such as floral organ differentiation and development regulation, energy metabolism and carbohydrates biosynthesis, which results in a partial anther exsertion and pollen fertility restoration in the partially rescued plants. The single nucleotide polymorphisms (SNPs) identified in two potential associated genes were validated to be related to the fertility restoration phenotype by KASP marker assays. This novel knowledge contributes to the understanding of the molecular mechanism of the partial fertility restoration of CMS-S in maize and thus helps to guide the breeding programs.</p></div

Paraffin slides show normal microspore development in the partially rescued FIL-B.

<p>Microscopic images of anther transverse sections of Jing724 (<b>A</b>) and the partially rescued (<b>B</b>) and sterile (<b>C</b>) individuals of FIL-B. The black round microspores were considered as normal. The scale bars represent 100 μm.</p

Partially restored anther exsertion in the FIL-B population.

<p><b>A.</b> Representative image of the tassels of Jing724 and FIL-B individuals with partial anther exsertion and complete sterility at six days after tasseling. The scale bars represent 3 cm. <b>B.</b> Representative image of the anthers harvested from A. The scale bars represent 3 cm.</p

Comparison of the transcript levels of the six potential associated genes from the partially rescued (F) and sterile (S) plants of FIL-B, as detected by QPCR.

<p>Each bar represents the mean±SE of the biological replicates. The values are calculated using Actin as an internal control. The asterisks show the statistically significant difference compared to the partially rescued plants, as determined by the analysis of variance: * (P<0.05), **(P<0.01), ***(P<0.001).</p

The physical position and number of genes in the five identified genomic regions on chromosome 2.

<p>The physical position and number of genes in the five identified genomic regions on chromosome 2.</p

SNPs in genes potentially associated with fertility instability identified from RNA-Seq

<p>SNPs in genes potentially associated with fertility instability identified from RNA-Seq</p

Enrichment of the KEGG pathways in the differentially expressed genes.

<p>Enrichment of the KEGG pathways in the differentially expressed genes.</p