Immediate premeiotic transcriptomic effects following nonchemically induced whole genome duplication in the moss Funaria hygrometrica

Abstract

Whole genome duplications (WGDs) have shaped the evolutionary history of land plants by driving innovation and diversification (One Thousand Plant Transcriptomes Initiative, 2019; Heslop-Harrison et al., 2023). Following such events, whether through genome doubling (autopolyploidy) or hybridization-driven merging (allopolyploidy), both DNA and histone methylation are activated. This activation triggers a rapid cascade of retroelement suppression and subsequent modifications to gene expression. Immediately following WGD, the total number of genes should be preserved (Birchler & Veitia, 2014; Doyle & Coate, 2019) such that shifts in expression would then be governed only by epigenetic processes. In studies examining gene dosage in autopolyploids (Yu et al., 2010; Coate et al., 2020; Song et al., 2020a), genome doubling is frequently induced via colchicine treatment, which itself may influence transcriptomic outcomes (Münzbergová, 2017). Additionally, these plants are typically assessed after at least one reproductive cycle, during which meiotic events could further shape gene expression. Genome doubling occurs naturally during key life cycle transitions, in the transition from haploid gametophyte to diploid sporophyte, and is associated with methylation changes that can modify or reset gene expression (Schmid et al., 2018; Rensing et al., 2020; Borg et al., 2021; Rempfer et al., 2022). However, it remains unclear whether a WGD event triggered by a homoploid shift from sporophyte to gametophyte, known as apospory, can induce gene expression changes in the absence of chemical induction and before the epigenetic reprogramming associated with meiosis.

Mosses represent excellent model systems for investigating the immediate transcriptomic effects of WGD for several reasons. First, their life cycle includes two macroscopic, easily manipulable generations: a haploid vegetative gametophyte and a diploid sporophyte, allowing direct comparisons between ploidy levels. Second, isogenic diploid gametophytes can be reliably induced from immature diploid sporophytes through apospory (Bryan, 2001), avoiding the confounding effects associated with chemical polyploidization methods such as a colchicine treatment (Münzbergová, 2017; Zhou et al., 2017). And third, unlike in grass models, in which diploid apospory is rare and inconsistently expressed (Ortiz et al., 2013), mosses provide a genetically stable and reproducible system for generating polyploid tissues. Additionally, their relatively simple body plan and compact genomes reduce the complexity of downstream transcriptomic analyses, making them an ideal system for studying the direct molecular consequences of WGD. Finally, gene expression in these aposporous diploid gametophytes (ADG, 2n) can be characterized immediately, that is before syngamy and meiosis resetting genetic programming.

Such moss experimental systems thus allow for the assessment of the transcriptomic impact of shifts in ploidy (e.g. haploid/diploid) vs in function (gametophyte/sporophyte) immediately following WGD in plants. Here, independent ADG (2n) are generated from sporophytes of the moss Funaria hygrometrica and their gene expression is contrasted against that of replicate parental haploid gametophytes and stages of the diploid (2n) sporophyte (Fig. 1a; Supporting Information Methods S1) to assess the immediate transcriptomic consequences. The observed differential gene expression is discussed in the context of long terminal repeats (LTRs) and methylation site distributions.

Cultures of F. hygrometrica (Funariaceae, Budke 145, CONN) were established from spores of a single sporophyte. As in the confamilial Physcomitrium patens, self-fertilization and, to a lesser extent, outcrossing likely drive sporophyte production in F. hygrometrica (Perroud et al., 2011), and it is assumed that the original sporophyte emerged from self-fertilization, was thus homozygous, and yielded isogenic haploid spores. Gametophytes were cultured and propagated under standard conditions and induced to undergo sexual reproduction through cold treatment triggering gametangia formation and gametogenesis. Immature sporophytes were sampled and cut in half, and the top of the premeiotic unswollen young sporophyte was placed on Knops medium. RNA was extracted from three replicates of vegetative haploid and diploid cultures and four developmental premeiotic stages of the sporophyte. A total of 10 RNA libraries (representing three stages) were sequenced on an Illumina NextSeq 500, and the resulting reads were aligned to the annotated chromosome-scale reference for F. hygrometrica (Kirbis et al., 2025). Repeat elements were independently classified from the reference, and cytosine methylation patterns were quantified from the genomic nanopore reads in all three contexts: CG, CHG, and CHH (where H stands for A, T, or C). Extended methods are available in Methods S1.

Inducing a WGD via apospory triggers a significant, immediate, and largely consistent transcriptomic response in the moss F. hygrometrica. In the first aposporous diploid (2n) gametophytes, 14% of the total gene space exhibits a shift in expression, and the majority of these genes (i.e. 7% or 10% of total) are consistently expressed or not expressed vs in the haploid (n) gametophytes. The transcriptomic profile of aposporous diploid (2n) gametophytes combines not only the expression of genes otherwise specific to the wild sporophyte (2n) or gametophyte (n) but also a suite of genes not expressed in the wild generations, that is it holds signatures linked to ploidy and function as well as de novo expressions. Whole genome duplication thus immediately generates new transcriptomic profiles for selection to act on, thereby potentially catalyzing rapid divergence rather than merely enabling subsequent radiation (Schranz et al., 2012) or escape from extinction (Van de Peer et al., 2021). Shifts in expression following WGD are largely governed by specific processes, given their repeatability across replicates, and likely involve changes in methylation in or near gene bodies and their associated LTRs, epigenetic processes known to largely shape plant development (Lucibelli et al., 2022). Contrasting the methylation landscapes of haploid (n) gametophytes, isogenic diploid (2n) sporophytes, and their aposporously derived diploid (2n) gametophytes across multiple generations will provide insights into the specific molecular mechanisms contributing to the observed gene expression profiles.

None declared.

BG and JLW designed the research. NR, VSV and NP generated and analyzed the data. YL and PS contributed data. BG wrote the first draft of the paper. All authors contributed to the final version. NP and VSV contributed equally to this work.

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