The mode of inheritance of the male sterility trait is crucial for understanding the evolutionary dynamics of the sexual system gynodioecy, which is the co-occurrence of female and hermaphrodite plants in natural populations. Both cytoplasmic (CMS) and nuclear (restorer) genes are known to be involved. Theoretical models usually assume a limited number of CMS genes with each a single restorer gene, while reality is more complex. In this study, it is shown that in the gynodioecious species Plantago coronopus two new CMS-restorer polymorphisms exist in addition to the two that were already known, which means four CMS-restorer systems at the species level. Furthermore, three CMS types were shown to co-occur within a single population. All new CMS types showed a multilocus system for male fertility restoration, in which both recessive and dominant restorer alleles occur. Our finding of more than two co-occurring CMS-restorer systems each with multiple restorer genes raises the question how this complex of male sterility systems is maintained in natural populations.

Environmentally induced DNA methylation variants may mediate gene expression responses to environmental changes. If such induced variants are transgenerationally stable, there is potential for expression responses to persist over multiple generations. Our current knowledge in plants, however, is almost exclusively based on studies conducted in sexually reproducing species where the majority of DNA methylation changes are subject to resetting in germlines, limiting the potential for transgenerational epigenetics stress memory. Asexual reproduction circumvents germlines, and may therefore be more conducive to long-term inheritance of epigenetic marks. Taking advantage of the rapid clonal reproduction of the common duckweed Lemna minor, we hypothesize that long-term, transgenerational stress memory from exposure to high temperature can be detected in DNA methylation profiles. Using a reduced representation bisulphite sequencing approach (epiGBS), we show that temperature stress induces DNA hypermethylation at many CG and CHG cytosine contexts but not CHH. Additionally, differential methylation in CHG context that was observed was still detected in a subset of cytosines, even after 3-12 generations of culturing in a common environment. This demonstrates a memory effect of stress reflected in the methylome and that persists over multiple clonal generations. Structural annotation revealed that this memory effect in CHG methylation was enriched in transposable elements. The observed epigenetic stress memory is probably caused by stable transgenerational persistence of temperature-induced DNA methylation variants across clonal generations. To the extent that such epigenetic memory has functional consequences for gene expression and phenotypes, this result suggests potential for long-term modulation of stress responses in asexual plants.

Background 5-Methylcytosine (5mC) is an important epigenetic mark in eukaryotes. Little information about its role exists for invertebrates. To investigate the contribution of 5mC to phenotypic variation in invertebrates, alteration of methylation patterns needs to be produced. Here, we apply new non-nucleoside DNA methyltransferase inhibitors (DNMTi) to introduce aleatory changes into the methylome of mollusk species. Results Flavanone inhibitor Flv1 was efficient in reducing 5mC in the freshwater snails Biomphalaria glabrata and Physa acuta, and to a lesser degree, probably due to lower stability in sea water, in the oyster Crassostrea gigas. Flv1 has no toxic effects and significantly decreased the 5mC level in the treated B. glabrata and in its offspring. Drug treatment triggers significant variation in the shell height in both generations. A reduced representation bisulfite-sequencing method called epiGBS corroborates hypomethylation effect of Flv1 in both B. glabrata generations and identifies seven Differential Methylated Regions (DMR) out of 32 found both in Flv1-exposed snails and its progeny, from which 5 were hypomethylated, demonstrating a multigenerational effect. By targeted bisulfite sequencing, we confirmed hypomethylation in a locus and show that it is associated with reduced gene expression. Conclusions Flv1 is a new and efficient DNMTi that can be used to induce transient and heritable modifications of the epigenetic landscape and phenotypic traits in mollusks, a phylum of the invertebrates in which epigenetics is understudied.

Eleven polymorphic microsatellite loci were obtained from a GA enriched genomic library, constructed from DNA of buck’s‐horn plantain (Plantago coronopus). The microsatellite loci were tested on 24 genotypes. These plants were collected from meadows along the coast, located on 11 sites ranging from the southwest to the northeast of the Netherlands. In this set of plants the isolated microsatellites were highly polymorphic with 3–24 alleles per locus and a maximum observed heterozygosity of 0.91. Some of the microsatellite loci also showed amplification in two other plantain species (P. lanceolata and P. maritima).

While some DNA methylation variants are transgenerationally stable in plants, DNA methylation modifications that are specifically induced by environmental exposure are typically transient and subject to resetting in germ lines, limiting the potential for transgenerational epigenetics stress memory. Asexual reproduction circumvents germlines, and may be more conducive to long-term memory and inheritance of epigenetic marks. This, however, has been poorly explored. Taking advantage of the rapid clonal reproduction of the common duckweed Lemna minor, we tested the hypothesis that a long-term, transgenerational stress memory from exposure to high temperature can be detected in DNA methylation profiles. Using a reduced representation bisulfite sequencing approach (epiGBS), we show that high temperature stress induces DNA hypermethylation at many cytosines in CG and CHG contexts but not in CHH. In addition, a subset of the temperature responsive CHG cytosines, showed differential DNA methylation between in lineages exposed to 30C and 24C, 3-12 clonal generations after subsequent culturing in a common environment, demonstrating a memory effect of stress that persists over many clonal generations and that is reflected in DNA methylation. Structural annotation revealed that this memory effect in CHG methylation was enriched in TEs. We argue that the observed epigenetic stress memory is likely caused by stable transgenerational persistence of high temperature-induced DNA methylation variants across multiple clonal generations. To the extent that such epigenetic memory has functional consequences for gene expression and phenotypes, this result suggests potential for long-term modulation of stress responses in asexual plants and vegetatively propagated crops.