De novo transcriptome assembly from flower buds of dioecious, gynomonoecious and chemically masculinized female Coccinia grandis reveals genes associated with sex expression and modification

Abstract

Background: Coccinia grandis (ivy gourd), is a dioecious member of Cucurbitaceae having heteromorphic sex chromosomes. Chromosome constitution of male and female plants of C. grandis is 22A + XY and 22A + XX respectively. Earlier we showed that a unique gynomonoecious form of C. grandis (22A + XX) also exists in nature bearing morphologically hermaphrodite flowers (GyM-H). Additionally, application of silver nitrate (AgNO₃) on female plants induces stamen development leading to the formation of morphologically hermaphrodite flowers (Ag-H) despite the absence of Y-chromosome. Due to the unavailability of genome sequence and the slow pace at which sex-linked genes are identified, sex expression and modification in C. grandis are not well understood.

Results: We have carried out a comprehensive RNA-Seq study from early-staged male, female, GyM-H, and Ag-H as well as middle-staged male and GyM-H flower buds. A de novo transcriptome was assembled using Trinity and annotated by BLAST2GO and Trinotate pipelines. The assembled transcriptome consisted of 467,233 'Trinity Transcripts' clustering into 378,860 'Trinity Genes'. Female_Early_vs_Male_Early, Ag_Early_vs_Female_Early, and GyM-H_Middle_vs_Male_Middle comparisons exhibited 35,694, 3574, and 14,954 differentially expressed transcripts respectively. Further, qRT-PCR analysis of selected candidate genes validated digital gene expression profiling results. Interestingly, ethylene response-related genes were found to be upregulated in female buds compared to male buds. Also, we observed that AgNO₃ treatment suppressed ethylene responses in Ag-H flowers by downregulation of ethylene-responsive transcription factors leading to stamen development. Further, GO terms related to stamen development were enriched in early-staged male, GyM-H, and Ag-H buds compared to female buds supporting the fact that stamen growth gets arrested in female flowers.

Conclusions: Suppression of ethylene responses in both male and Ag-H compared to female buds suggests a probable role of ethylene in stamen suppression similar to monoecious cucurbits such as melon and cucumber. Also, pollen fertility associated GO terms were depleted in middle-staged GyM-H buds compared to male buds indicating the necessity of Y-chromosome for pollen fertility. Overall, this study would enable identification of new sex-biased genes for further investigation of stamen arrest, pollen fertility, and AgNO₃-mediated sex modification.

Keywords: Coccinia grandis; De novo transcriptome; Dioecious; Ethylene; Gynomonoecious; Pollen fertility; Silver nitrate; Stamen arrest.

Fig. 1

Different stages of C. grandis flower buds selected for RNA-Seq analysis. (a) Male, (b) Female flower buds. GyM-H and Ag-H buds sized similar to female buds were chosen for RNA seq study as described in our previous work (Ghadge et al., 2014). Scale bar = 1 cm

Fig. 2

Transcript size distribution for C. grandis flower bud transcriptome

Fig. 3

BLAST2GO annotation of C. grandis flower bud transcriptome. (a) BLAST Top-Hits species distribution when compared with Swiss-Prot database, (b) BLAST Top-Hits species distribution when compared with nr database, (c) GO category distribution of C. grandis flower bud transcriptome

Fig. 4

Correlation analyses showing the relationship between samples and replicates. (a) Principal component analysis and (b) correlation matrix showing relationship between all samples as well as replicates

Fig. 5

Pairwise comparisons of transcript abundance. MA plots showing average log fold change (logFC) vs average log of counts among (a) female (early-staged) vs. male (early-staged) transcripts, (b) female (early-staged) vs. GyM-H (early-staged) transcripts, (c) Ag-H (early-staged) vs. female (early-staged) transcripts and (d) GyM-H (middle-staged) vs. male (middle-staged) across replicates. Volcano plots showing differentially expressed transcripts in relation to FDR (False discovery rate) for (e) female (early-staged) vs. male (early-staged) transcripts, (f) female (early-staged) vs. GyM-H (early-staged) transcripts, (g) Ag-H (early-staged) vs. female (early-staged) transcripts and (h) GyM-H (middle-staged) vs. male (middle-staged). Features found DE at FDR <0.05 are colored red. Features with P-values at most 1e-3 and at least 2^2 fold change are differentially expressed

Fig. 6

Hierarchical clustering of differentially expressed transcripts and developmentally staged C. grandis flower bud samples. Heatmap showing the relative expression levels of each transcript (rows) in each sample (columns). Rows and columns are hierarchically clustered. Expression values (FPKM) are log₂ –transformed and then median-centered by transcript

Fig. 7

Validation of selected DE genes by qRT-PCR with two biological replicates. The relative expression in the sample of middle-staged male-A (MMA) was set to 1 for plotting the qRT-PCR data. AgEA, Early-staged Ag-H A; AgEB, Early-staged Ag-H B; FEA, Early-staged Female A; FEB, Early-staged Female B; GEA, Early-staged GyM-H A; GEB, Early-staged GyM-H B; GMA, Middle-staged GyM-H A; GMB, Middle-staged GyM-H B; MEA, Early-staged Male A; MEB, Early-staged Male B; MMA, Middle-staged Male A; MMB, Middle-staged Male B; AgMA, Middle-staged Ag-H A, AgMB, Middle-staged Ag-H B; FMA, Middle-staged Female A; FMB, Middle-staged Female B

Fig. 8

qRT-PCR based expression analyses of selected genes involved in pollen tube development with two biological replicates. The relative expression in the sample of middle-staged male-A (MMA) was set to 1 for plotting the qRT-PCR data. AgEA, Early-staged Ag-H A; AgEB, Early-staged Ag-H B; FEA, Early-staged Female A; FEB, Early-staged Female B; GEA, Early-staged GyM-H A; GEB, Early-staged GyM-H B; GMA, Middle-staged GyM-H A; GMB, Middle-staged GyM-H B; MEA, Early-staged Male A; MEB, Early-staged Male B; MMA, Middle-staged Male A; MMB, Middle-staged Male B; AgMA, Middle-staged Ag-H A, AgMB, Middle-staged Ag-H B; FMA, Middle-staged Female A; FMB, Middle-staged Female B

Fig. 9

qRT-PCR based expression analyses of selected Ethylene-responsive transcription factors (ERFs) with two biological replicates. The relative expression in the sample of middle-staged male-A (MMA) was set to 1 for plotting the qRT-PCR data. AgEA, Early-staged Ag-H A; AgEB, Early-staged Ag-H B; FEA, Early-staged Female A; FEB, Early-staged Female B; GEA, Early-staged GyM-H A; GEB, Early-staged GyM-H B; GMA, Middle-staged GyM-H A; GMB, Middle-staged GyM-H B; MEA, Early-staged Male A; MEB, Early-staged Male B; MMA, Middle-staged Male A; MMB, Middle-staged Male B; AgMA, Middle-staged Ag-H A, AgMB, Middle-staged Ag-H B; FMA, Middle-staged Female A; FMB, Middle-staged Female B