Fig 1.
. Morphological characteristics of the male sterile and male fertile two-type line M525AB of T. erecta.
(a) Plant morphology of male sterile plant M525A (right) and male fertile plant M525B (left); (b) Inflorescence morphology of male fertile plant M525B; (c) Inflorescence morphology of male sterile plant M525A. RF: ray floret, DF: disc floret, SF: sterile floret.
Fig 2.
Floret morphology of the male sterile and male fertile two-type line M525AB of T. erecta.
The ray florets of male sterile M525A (a-1) and male fertile M525B (b-1) had three whorls of floral organs, sepal (a-2, b-2), petal (a-3, b-3) and pistil (a-4, b-4), while the petal of ray floret in M525A developed into sepal-like structure. The disk florets of male sterile M525A (c-1) and male fertile M525B (d-1) had four whorls of floral organs, sepal (c-2, d-2), petal (c-3, d-3), stamen (c-5, d-5) and pistil (c-4, d-5). The petals of disc florets in M525A developed into sepal-like structures, while the stamens developed into yellow filaments.
Fig 3.
Scanning electron microscope observation of floret morphology of the male sterile and male fertile two-type line M525AB of T. erecta.
The deformed petal of male sterile plant was covered by unusual pappus hairs which were typically found in sepal. The distorted stamen of male sterile plant was covered by trichomes that were only found in stigma walls. Pa: pappus hairs, Tr: trichomes.
Fig 4.
Flower buds development processes of male sterile and male fertile two-type line M525AB of T. erecta.
(a) Developmental process of male sterile M525A’s flower buds from 0.5 mm to 8 mm in diameter; (b) Developmental process of male fertile M525B’s flower buds from 0.5 mm to 8 mm in diameter. IP: inflorescence primordium, SFP: sterile floret primordium, SF: sterile floret, SP: sepal and sepal-like petal of male sterile floret, SS: style-like stamen and stigma of male sterile floret, RFP: ray floret primordium, DFP: disc floret primordium, RF: ray floret, DF: disc floret, Se: sepal of fertile floret, Pe: petal of ray floret, St: stigma of ray floret.
Table 1.
Number of unigenes annotated in databases.
Fig 5.
Functional classifications of the assembled unigenes according to the euKaryotic Ortholog Group categories.
The x-axis indicated 26 groups of KOG. The y-axis indicated the percentage of the number of annotated genes under a group to the total number of annotated genes.
Fig 6.
Gene Ontology classifications of the assembled unigenes.
The results were categorized into three major categories: cellular component, molecular function, and biological process. The right y-axis indicated the number of genes in a category. The left y-axis indicated the percentage of a specific category of genes in that main category.
Fig 7.
Functional classification of KEGG pathway of assembled unigenes.
The KEGG pathways were summarized in five main categories: A, Cellular Processes; B, Environmental Information Processing; C, Genetic Information Processing; D, Metabolism; E, Organismal Systems. The y-axis indicated the name of the KEGG metabolic pathways. The x-axis indicated the percentage of the number of genes annotated under that pathway in the total number of annotated genes.
Fig 8.
Length distribution of CDS prediction and translation.
(a) The length distribution of the predicated CDS sequences using NCBI blast 2.2.28+; (b) The length distribution of the predicated amino acid sequences using NCBI blast 2.2.28+; (c) The length distribution of the predicated CDS sequences using Estscan (3.0.3) software; (d) The length distribution of the predicated amino acid sequences using Estscan (3.0.3) software.
Fig 9.
Differentially expressed genes (DEGs) of flower buds from male sterile and male fertile plants.
(a) DEGs between S1 (1 mm flower buds of male sterile plants) and F1(1 mm flower buds of male fertile plants); (b) DEGs between S2 (4 mm flower buds of male sterile plants) and F2 (4 mm flower buds of male fertile plants); (c) The Venn diagram showed specifically or commonly expressed DEGs in both development of flower buds. In the volcano figure, scattered dot represented each gene, blue dots indicated that the unigenes with no significant differential expression level, red dots indicated the significantly up-regulated unigenes while the green dots indicated the significantly down-regulated unigenes. In the Venn diagram, the number in the large circle represented total number of specifically expressed DEGs in 1 mm or 4 mm sized flower buds, while the number in the overlapping portion represented commonly expressed DEGs in both 1 mm and 4 mm sized flower buds.
Fig 10.
GO term enrichment analysis of differentially expressed genes of flower buds between male sterile and male fertile plants.
(a) Enriched GO term between S1 (1 mm flower buds of male sterile plants) and F1 (1 mm flower buds of male fertile plants); (b) Enriched GO term between S2 (4 mm flower buds of male sterile plants) and F2 (4 mm flower buds of male fertile plants). The results were categorized into three major categories (BP: biological process, CC: cellular component, MF: molecular function). The left y-axis represented the percentage of DEGs annotated in this term. The digits above the GO terms represented the number of DEGs annotated in this term (including the sub-term).
Fig 11.
Heat map diagram of expression levels of DEGs annotated in the MADS-box transcription.
Data for the relative expression levels of genes were obtained by DGE data after taking log10 (FPKM+1). Color from red to blue, indicated that the log10 (FPKM+1) values were from large to small, red color indicates high expression level and blue color indicates low expression level.
Table 2.
MADS-box unigenes showing significantly different expression between S2 and F2.
Fig 12.
qRT-PCR verifications of seven MADS-box genes.
The x axis represented four samples. S1: 1 mm flower buds of male sterile plants, F1: 1 mm flower buds of male fertile plants, S2: 4 mm flower buds of male sterile plants, F2: 4 mm flower buds of male fertile plants. The Left y axis represented the relative expression level by qRT-PCR. The right y axis is the FPKM value by DGE analysis.
Fig 13.
Linear regression analysis of the fold change of the gene expression ratios between DEG sequencing and qRT-PCR.
26 unigenes were selected for quantitative real-time PCR analysis to confirm the accuracy and reproducibility of the Illumina expression profiles using the same RNA samples that were used for DGE sequencing. The relative expression levels of the genes were calculated using the 2−ΔΔCt method in qRT-PCR analysis. The DGE sequencing data were represented by the FPKM value of samples. Scatterplots were generated by the log2 expression ratios from DGE sequencing data (x-axis) and qRT-PCR data (y-axis).