Comparative RNA-Seq analysis reveals genes associated with masculinization in female Cannabis sativa

Abstract

Using RNA profiling, we identified several silver thiosulfate-induced genes that potentially control the masculinization of female Cannabis sativa plants. Genetically female Cannabis sativa plants normally bear female flowers, but can develop male flowers in response to environmental and developmental cues. In an attempt to elucidate the molecular elements responsible for sex expression in C. sativa plants, we developed genetically female lines producing both female and chemically-induced male flowers. Furthermore, we carried out RNA-Seq assays aimed at identifying differentially expressed genes responsible for male flower development in female plants. The results revealed over 10,500 differentially expressed genes, of which around 200 potentially control masculinization of female cannabis plants. These genes include transcription factors and other genes involved in male organ (i.e., anther and pollen) development, as well as genes involved in phytohormone signalling and male-biased phenotypes. The expressions of 15 of these genes were further validated by qPCR assay confirming similar expression patterns to that of RNA-Seq data. These genes would be useful for understanding predisposed plants producing flowers of both sex types in the same plant, and help breeders to regulate the masculinization of female plants through targeted breeding and plant biotechnology.

Keywords: Cannabis; Differential expression; Flower sex; Male biased-genes; RNA-seq; Silver thiosulfate.

Fig. 1

Floral bud samples of cannabis plants used for RNA-Seq studies between flower sex types. The flower sex types are normal male flower buds from genetically male plants; Ag₂S₂O₃-induced male flower buds from genetically female plants; and normal female flower buds from genetically female plants. These flower buds were collected for RNA isolation at 14 days of post-light and fertigation induction of flowering

Fig. 2

Hierarchical clustering of DEGs in different expression analysis assays. a IMFvsFF, b MFvsFF and c IMFvsMF libraries. Heatmaps show the relative expression levels of each transcript (row) in each flower sample (column). A comparable number of transcripts were differentially expressed in IMFvsFF (15%) and MFvsFF (17.6%) libraries, while slightly low numbers of DEGs (10%) were detected in IMFvsMF library, with the cut-off value of log₂FC ≥ │1│and FDR ≤ 0.05. Expression values were normalized by RPKM and expressed as log₂ fold change (log₂FC), with a cut-off value of ≥ │1│and FDR p value ≤ 0.05. The color scale indicates upregulation (red) and downregulation (blue) of the transcripts in the samples

Fig. 3

Expression of transcripts between cannabis floral sex types. Volcano plots displaying the expression of transcripts with false discovery rate (FDR) for a IMFvsFF, b MFvsFF, and c IMFvsMF. The log₁₀(p values) indicates FDR p values. Transcripts with log₂ fold change values of ≥ │1│ and FDR p value of ≤ 0.05 (–log₁₀(p value) ≥ 1.30) are differentially expressed between the flower sex types. d Venn diagram showing the number of DEGs between cannabis flower sex types. With the cut-off value of log₂FC ≥ │1│ and FDR ≤ 0.05, a total of 10,833 transcripts in IMFvsFF and of 12,986 transcripts in MFvsFF comparison were differentially expressed, from which nearly 50% of these DEGs were common in both comparisons. A total of 7007 transcripts were represented in the IMFvsMF comparison group, of which 793 (~ 11%) were differentially expressed only in this comparison

Fig. 4

Heatmaps of key DEGs potentially controlling sex determination and floral development in IMFvsFF, MFvsFF and IMFvsMF libraries. a DEGs linked to floral development and sex determination. From a total of 245 DEGs, 61 DEGs (25%) potentially controlled anther/pollen development, floral transition and floral organ identity were identified in the three comparisons. More DEGs are involved in male flower regulations than those in female flowers. b DEGs associated with major hormone pathways and signalling. A total of 50 DEGs was identified in three comparisons. These DEGs are distributed to abscisic acid (ABA), auxin, cytokinin, ethylene, and gibberellin signalling pathways. The majority of the genes were differentially expressed in IMFvsFF and MFvsFF, while a few (19 DEGs) were detected from IMFvsMF. The relative expression of each gene (row) in each comparison (column) is shown. Expression values are log₂ fold changes (log₂FC) with color scales of red (upregulated) and blue (downregulated). MFvsFF library was used as a control for IMFvsFF comparison

Fig. 5

qPCR validation of selected DEGs in flowers of silver thiosulfate-induced male (IMF) and normal female (FF) plants. These DEGs include nine transcription factor genes: APETALA 3 (AP3), Dysfunctional Tapetum1 (DYT1), Agamous-like MADS-box 11 (AGL11), MADS2, WUSCHEL (WUS), MYB35, MYB80, bHLH91 and ABORTED MICROSPORES (AMS), and six other genes: Spermidine hydroxycinnamoyl transferase (SHT), Eceriferum 26-like (CR26), MEN-8 (male-specific protein—Men8), Cytochrome P450 703A2 (C70A2), Serine threonine- kinase AFC2 (AFC2) and Mannose glucose-specific lectin (LEC) involved in floral development and sex determination. qPCR data were expressed as mean values ± standard errors (n = 2) of log₂ fold change. The relative expression (qPCR) in FF samples was set arbitrary to 1 (log₂(1) = 0). RNA-Seq data were shown as mean values ± standard errors (n = 2–4) of log₂ RPKM. All of the targeted genes had similar expression patterns in both qPCR and RNA-Seq analyses between the two flower sex types

Fig. 6

qPCR assays of selected DEGs in flowers of normal male (MF) and female (FF) plants. These DEGs include nine transcription factor genes: APETALA 3 (AP3), Dysfunctional Tapetum1 (DYT1), Agamous-like MADS-box 11 (AGL11), MADS2, WUSCHEL (WUS), MYB35, MYB80, bHLH91and ABORTED MICROSPORES (AMS), and six other genes: Spermidine hydroxycinnamoyl transferase (SHT), Eceriferum 26-like (CR26), MEN-8 (male-specific protein—Men8), Cytochrome P450 703A2 (C70A2), Serine threonine- kinase AFC2 (AFC2) and Mannose glucose-specific lectin (LEC) involved in floral development and sex determination. qPCR data were expressed as mean values ± standard errors (n = 2) of log₂ fold change. The relative expression (qPCR) in the samples of FF was set arbitrary to 1 (log₂(1) = 0). RNA-Seq data were shown as mean values ± standard errors (n = 2–4) of log₂ RPKM. All of the targeted genes had similar expression patterns in both qPCR and RNA-Seq analyses between the two flower sex types