Figure 1.
Flow cytometric seed screen on single seeds of Boechera gunnisoniana to analyse ploidy.
Fluorescence intensity was measured on individual green seeds of Boechera to determine the ploidy level of embryo and endosperm. 98% of the seeds measured (N = 84) showed a 3C∶9C ratio of embryo∶endosperm, indicating diplosporous apomixis (A). The remaining 2% showed a 6C∶9C embryo∶endosperm ratio indicative of a BIII hybrid where the embryo is derived from fertilization of an unreduced egg cell (B).
Figure 2.
Laser-assisted microdissection (LAM) and transcriptome analysis to study the Boechera apomictic initial cell (AIC).
(A, B) LAM of the AIC from a 6 µm dry section (scale bar = 40 µm). (A) An ovule harbouring the AIC before LAM. Arrows point to the AICs. (B) The ovule after the AIC has been dissected and collected. (C, D) Venn diagrams showing the overlaps of prediction of expression (P calls; apo_initial1, apo_initial2, MMC_M/P) as determined with the BgPANP algorithm or the AtPANP algorithm described previously [12], and the genes with ≥5 read counts on Boechera homologues (apo_initial3) to the Arabidopsis genes as determined by mapping to the Boechera reference transcriptome.
Table 1.
Transcriptome analysis of 11 samples from apomictic Boechera isolated by LAM.
Figure 3.
Independent data validation for selected genes by in situ hybridization on B. gunnisoniana ovules.
Data validation for selected genes found expressed in the Boechera AIC but not the Arabidopsis MMC. Scale bars are 20 µm, arrows point to the AICs. In situ hybridizations on B. gunnisoniana ovule sections were performed with antisense probes (A–C, E, F, H, I, K, M, N) or sense probes as controls (D, G, J, L, O) for the transcription factors AT1G06170, a basic helix-loop-helix (bHLH) DNA-binding superfamily protein (A–D), AT1G28050, a B-BOX DOMAIN PROTEIN 13 (E–G), and AT1G76580 a Squamosa promoter-binding protein-like (SBP domain) transcription factor family protein (H–J), an oligopeptide transporter, AT1G59740 (K,L), and AT1G14900, encoding the HIGH MOBILITY GROUP A protein.
Figure 4.
Heatmap of log2 transformed normalized read counts.
Heatmap of 1'487 genes enriched in the Boechera AIC as compared to all cell types of the mature female gametophyte as identified using NOISeq-sim (A). Heatmap of 3'792 genes enriched in the AIC as compared to all cell types of the mature gametophyte or differentially expressed between any cell type of the mature gametophyte identified using EdgeR (B). The hierarchical clustering of samples and genes was based on euclidean distance and hierarchical agglomerative clustering. Colours are scaled per row and red denotes high expression and black low expression.
Table 2.
Gene ontology analysis.
Table 3.
Gene ontology analysis on MMC and AIC.
Table 4.
Gene ontology analysis on sexual and parthenogenetic egg cells of Arabidopsis and Boechera, respectively.
Table 5.
Analysis of PFAM domains significantly enriched in the Boechera germline.
Table 6.
Gene family enrichment.
Figure 5.
Heatmap of log2 transformed normalized read counts for 14 selected meiotic or MMC-expressed genes.
Hierachical clustering of read counts from different Arabidopsis and Boechera cell- and tissue types [13], [62]–[66]. The hierarchical clustering of samples and genes was based on euclidean distance and hierarchical agglomerative clustering. Colours are scaled per row. Red denotes high expression and black low expression.
Figure 6.
Analysis of sequence divergence of members of the AtRKD gene family.
Analysis of sequence divergence of members of the AtRKD gene family and close Boechera homologues as analysed with ClustalX based on protein sequences and read counts assigned. Boechera gene model variants are indicated with compxxx_cY_seqZ.
Figure 7.
Heatmap clustering of members of the AtRKD gene family.
Heatmap of normalized log2 transformed read counts from different Arabidopsis and Boechera cell- and tissue-types [13], [62]–[66]. The hierarchical clustering of samples and genes was based on euclidean distance and hierarchical agglomerative clustering. No row scaling of colours was applied. Red denotes high expression and black low expression.