Fig 1.
Partially restored anther exsertion in the FIL-B population.
A. 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. Representative image of the anthers harvested from A. The scale bars represent 3 cm.
Fig 2.
Pollen staining with I2-KI revealed the normal development of a small portion of pollen from the partially rescued FIL-B.
Representative images of the stained pollen of Jing724 (A) and the partially rescued (B) and sterile (C) 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.
Fig 3.
Paraffin slides show normal microspore development in the partially rescued FIL-B.
Microscopic images of anther transverse sections of Jing724 (A) and the partially rescued (B) and sterile (C) individuals of FIL-B. The black round microspores were considered as normal. The scale bars represent 100 μm.
Table 1.
Enrichment of the KEGG pathways in the differentially expressed genes.
Table 2.
The physical position and number of genes in the five identified genomic regions on chromosome 2.
Fig 4.
Identification of genomic regions contributing to the fertility instability in FIL-B by BSR-Seq using the Euclidean distance (ED) algorithm.
A. 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. The ED5 scores of a close-up of chromosome 2. C. The Loess fit curve calculated from A. D. 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.
Table 3.
Twelve genes with differential expression from the 5 genomic regions identified on chromosome 2.
Fig 5.
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.
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).
Fig 6.
Graphs of four KASP marker assays.
KASP assays were developed for SNPs identified in two potential associated genes from RNA-Seq. Fertility instable line (FIL)-1 and FIL-2 were SNPs resided in GRMZM2G315401. FIL-3 and FIL-4 were SNPs detected in GRMZM2G430362. The triangles represent the recurrent parent of Jing724. Black data points are negative control and pink ones are ambiguous calling.
Table 4.
SNPs in genes potentially associated with fertility instability identified from RNA-Seq