The identification of differentially expressed genes in male and female gametophytes of simple thalloid liverwort Pellia endiviifolia sp. B using an RNA-seq approach

Abstract

This study shows differences in gene expression between male and female gametophytes of the simple thalloid liverwort with a distinction between the vegetative and reproductive phases of growth. Pellia endiviifolia is a simple thalloid liverwort that, together with hornworts and mosses, represents the oldest living land plants. The limited taxon sampling for genomic and functional studies hampers our understanding of processes governing evolution of these plants. RNA sequencing represents an attractive way to elucidate the molecular mechanisms of non-model species development. In the present study, RNA-seq was used to profile the differences in gene expression between P. endiviifolia male and female gametophytes, with a distinction between the vegetative and reproductive phases of growth. By comparison of the gene expression profiles from individuals producing sex organs with the remaining thalli types, we have determined a set of genes whose expression might be important for the development of P. endiviifolia reproductive organs. The selected differentially expressed genes (DEGs) were categorized into five main pathways: metabolism, genetic information processing, environmental information processing, cellular processes, and organismal systems. A comparison of the obtained data with the Marchantia polymorpha transcriptome resulted in the identification of genes exhibiting a similar expression pattern during the reproductive phase of growth between members of the two distinct liverwort classes. The common expression profile of 87 selected genes suggests a common mechanism governing sex organ development in both liverwort species. The obtained RNA-seq results were confirmed by RT-qPCR for the DEGs with the highest differences in expression level. Five Pellia-female-specific and two Pellia-male-specific DEGs showed enriched expression in archegonia and antheridia, respectively. The identified genes are promising candidates for functional studies of their involvement in liverwort sexual reproduction.

Keywords: Antheridia; Archegonia; Bryophytes; Generative phase; Thalli; Vegetative phase.

Fig. 1

Chart listing statistics of the groups of differentially expressed genes between the two P. endiviifolia thalli comparison for DEGs showing similarity to sequences deposited in the public databases. The differentially expressed genes were identified with the criteria (twofold or more change and filtered using edgeR tool with FDR ≤ 0.05). In a pairwise comparison, the former one is considered as the control, and the latter one is considered as the treatment. Fiv female thalli grown in vitro, Fng female thalli with archegonia collected from natural habitat, Miv male thalli grown in vitro, Mng male thalli with antheridia collected from natural habitat

Fig. 2

Comparison of gene expression data obtained by RNA-seq and RT-qPCR. Expression levels of selected genes in four types of Pellia thalli are shown. Fiv female thalli grown in vitro, Fng female thalli with archegonia collected from natural habitat, Miv male thalli grown in vitro, Mng male thalli with antheridia collected from natural habitat. Y-axis on the left side of graphs shows the scale for qPCR values (grey boxes) normalized against ACTIN1 gene from two biological replicates. Y-axis on the right side of graphs shows the scale for RPKM values (black squares) from NGS

Fig. 3

Several P. endiviifolia genes expression is correlated with antheridia or archegonia production. Quantitative RT-qPCR analyses of the (a) male-specifically expressed RDA-cDNA/RNA-seq identified genes and (b) the female-specifically expressed RNA-seq identified genes with different transcript levels in the vegetative and sex organs of male and female gametophytes grown in natural habitat, respectively. a The relative expression of each gene was compared with antheridia (Mng.anth) isolated from the vegetative parts of male thalli (Mng.veg). b The relative expression of each gene was compared with archegonia-bearing region (Fng.arch) isolated from the vegetative parts of female thalli (Fng.veg). All transcript levels were normalized against ACTIN1. Error bars indicate mean ± SD; n = 2