Pearson bivariate correlation analysis of RNA-seq (log10) versus qPCR (log10) results.

Pearson bivariate correlation analysis of RNA-seq (log10) versus qPCR (log10) results.</p

Fig 7 -

Relative expression of MADS-box (A-D), fruit development and ovule, and carpel development (E-I), and lipid biosynthesis (J-M) genes from qPCR analysis in one week after pollination (1 WAP) and three weeks after pollination (3 WAP) fruit valves of E. pusilla. The x-axis shows the two samples of 1 WAP and 3 WAP. The y-axis represents the relative expression (in log2) of genes as the mean of three biological replicates’ expression and normalized relative to floral bud tissues. The asterisks denote statistically significant differences in the relative expression of the various developmental stages (*p≤  0.05).</p

Accession number of <i>rbc</i>L, <i>mat</i>K, nrITS sequences of orchid species used for ancestral state reconstruction analyses.

Accession number of rbcL, matK, nrITS sequences of orchid species used for ancestral state reconstruction analyses.</p

Summary of expression patterns assessed by RNA-seq and qPCR of genes involved in <i>Erycina pusilla</i> fruit development.

Expression of EpRPL gene is higher (indicated in the purple triangle) in 1 WAP fruits but lower in 3 WAP fruits. Expression of EpBs, EpKCS6, EpGL1, EpPDH-E1, and EpMAH1 genes is higher (indicated in the yellow triangle) in 3 WAP fruits (right side) but lower in the 1 WAP fruits. Expression of EpAG2, EpAGL6-2, EpSEP3, EpKOR1, EpSLR1, EpCRC/DL1, and EpTPL genes is high (indicated by the white box) in the 1 WAP and 3 WAP fruits.</p

Orchid fruit traits scoring.

Fruits play a crucial role in seed dispersal. They open along dehiscence zones. Fruit dehiscence zone formation has been intensively studied in Arabidopsis thaliana. However, little is known about the mechanisms and genes involved in the formation of fruit dehiscence zones in species outside the Brassicaceae. The dehiscence zone of A. thaliana contains a lignified layer, while dehiscence zone tissues of the emerging orchid model Erycina pusilla include a lipid layer. Here we present an analysis of evolution and development of fruit dehiscence zones in orchids. We performed ancestral state reconstructions across the five orchid subfamilies to study the evolution of selected fruit traits and explored dehiscence zone developmental genes using RNA-seq and qPCR. We found that erect dehiscent fruits with non-lignified dehiscence zones and a short ripening period are ancestral characters in orchids. Lignified dehiscence zones in orchid fruits evolved multiple times from non-lignified zones. Furthermore, we carried out gene expression analysis of tissues from different developmental stages of E. pusilla fruits. We found that fruit dehiscence genes from the MADS-box gene family and other important regulators in E. pusilla differed in their expression pattern from their homologs in A. thaliana. This suggests that the current A. thaliana fruit dehiscence model requires adjustment for orchids. Additionally, we discovered that homologs of A. thaliana genes involved in the development of carpel, gynoecium and ovules, and genes involved in lipid biosynthesis were expressed in the fruit valves of E. pusilla, implying that these genes may play a novel role in formation of dehiscence zone tissues in orchids. Future functional analysis of developmental regulators, lipid identification and quantification can shed more light on lipid-layer based dehiscence of orchid fruits.</div

Fig 2 -

Ancestral state reconstruction of fruit ripening period (A), fruit orientation (B), fruit dehiscence type (C), number of slits in mature fruit (D), fusion of valves (E), and lignification of dehiscence zone (F) characters from stochastic mapping analysis based on joint sampling of nrITS, matK and rbcL (10,000 mapped trees). Posterior probabilities (pie charts) are mapped in a random stochastic character map. Note: * nodes have bootstrap values A-B) Character 1: Erycina pusilla (ripe in ≤4 months), Phalaenopsis equestris (ripe in >4 months); character 2: Habenaria eustachys (erect), Papilionanthe teres (pendant). (C-D) Character 3: Vanilla pompona (indehiscent), Arundina graminifolia (dehiscent); character 4: Lockhartia acuta (Guarianthe skinneri (≥3 slits). (E-F) Character 5: A. graminifolia (valves fused at the base and apex), Ornithocephalus valerioi (valves fused at the base only); character 6: E. pusilla (non-lignified dehiscence zones), Cynorkis fastigiata (lignified dehiscence zones).</p

RNA-seq heatmap (TPM) of MICK-MADS-box transcription factors transcriptionally active in one week after pollination (1 WAP) and three weeks after pollination (3 WAP) fruits of <i>E</i>. <i>pusilla</i>.

RNA-seq heatmap (TPM) of MICK-MADS-box transcription factors transcriptionally active in one week after pollination (1 WAP) and three weeks after pollination (3 WAP) fruits of E. pusilla.</p

RNA-seq heatmap (TPM) of carpel, gynoecium, and ovule developmental genes transcriptionally active in one week after pollination (1 WAP) and three weeks after pollination (3 WAP) fruits of <i>E</i>. <i>pusilla</i>.

RNA-seq heatmap (TPM) of carpel, gynoecium, and ovule developmental genes transcriptionally active in one week after pollination (1 WAP) and three weeks after pollination (3 WAP) fruits of E. pusilla.</p

Fig 4 -

Ancestral state reconstructions of (A) character 2 (fruit orientation) vs 1 (ripening period), (B) character 4 (number of slits in mature fruit) vs 3 (dehiscence type, (C) character 5 (fusion of valves) vs character 2 (fruit orientation), (D) character 6 (lignified dehiscence zone) vs character 2 (fruit orientation), (E) character 4 (number of slits in mature fruit) vs character 3 (dehiscence type), and (F) character 5 (fusion of valves) vs character 6 (lignified dehiscence zone) from stochastic mapping analyses based on a joint sampling of nrITS, matK and rbcL (10000 map trees). The numbers represent the estimated number of evolutionary changes and the time spent in each state, arrows represent the direction of the transition between the states, and character combinations indicated with a square are interpreted as having co-evolved.</p

Changes in gene expression profile of 1 WAP and 3 WAP fruits of <i>Erycina pusilla</i>.

Upregulated genes are shown in red, while the number of downregulated genes is shown in blue.</p