Mimulus sRNAs Are Wound Responsive and Associated with Transgenerationally Plastic Genes but Rarely Both

Organisms alter development in response to environmental cues. Recent studies demonstrate that they can transmit this plasticity to progeny. While the phenotypic and transcriptomic evidence for this “transgenerational plasticity” has accumulated, genetic and developmental mechanisms remain unclear. Plant defenses, gene expression and DNA methylation are modified as an outcome of parental wounding in Mimulus guttatus. Here, we sequenced M. guttatus small RNAs (sRNA) to test their possible role in mediating transgenerational plasticity. We sequenced sRNA populations of leaf-wounded and control plants at 1 h and 72 h after damage and from progeny of wounded and control parents. This allowed us to test three components of an a priori model of sRNA mediated transgenerational plasticity—(1) A subset of sRNAs will be differentially expressed in response to wounding, (2) these will be associated with previously identified differentially expressed genes and differentially methylated regions and (3) changes in sRNA abundance in wounded plants will be predictive of sRNA abundance, DNA methylation, and/or gene expression shifts in the following generation. Supporting (1) and (2), we found significantly different sRNA abundances in wounded leaves; the majority were associated with tRNA fragments (tRFs) rather than small-interfering RNAs (siRNA). However, siRNAs responding to leaf wounding point to Jasmonic Acid mediated responses in this system. We found that different sRNA classes were associated with regions of the genome previously found to be differentially expressed or methylated in progeny of wounded plants. Evidence for (3) was mixed. We found that non-dicer sRNAs with increased abundance in response to wounding tended to be nearby genes with decreased expression in the next generation. Counter to expectations, we did not find that siRNA responses to wounding were associated with gene expression or methylation changes in the next generation and within plant and transgenerational sRNA plasticity were negatively correlated

The Molecular and Evolutionary Outcomes of Transgenerational Plasticity in Mimulus guttatus

Derived from the same family line, in the same greenhouse, and self-pollinated in an identical fashion, what effect could hole-puncher induced leaf damage have on the offspring of these individuals? Across three independent experiments during my graduate career I have demonstrated the diverse array of lingering epigenetic, gene expression, phenotypic, and fitness effects that simple mechanical wounding has on the following generation. While focused initially on the epigenetic and gene expression basis of increased trichome density in the offspring of damaged plants, it became clear early on that a host of other pathways were also differentially regulated. In the end I identified hundreds of differentially expressed genes and thousands of genomic regions where DNA methylation varied depending on parental wounding. Along with identifying numerous differentially regulated hormone synthesis genes and secondary metabolism pathways, I twice confirmed the differential regulation of the previously identified transgenerationally plastic Mimulus guttatus MYB Mixta-like 8 (MgMYBML8). A brief foray into molecular epigenetics and gene expression modeling provided our lab with the necessary information to utilize DNA methylation data and test hypotheses regarding the role of DNA methylation on transgenerational inheritance. Finally, through the use of a two-generation common garden experiment I demonstrated that transgenerational effects alter the development and resistance of plants in nature. While much remains to be deciphered regarding the molecular underpinnings, evolutionary role, and ecological relevance of transgenerational inheritance, the work presented herein provides a relatively comprehensive look at the complexity underlying an extremely simplified case of transgenerational inheritance

Parental experience modifies the Mimulus methylome

This work is licensed under a Creative Commons Attribution 4.0 International License.Background Transgenerational plasticity occurs when the environmental experience of an organism modifies the growth and development of its progeny. Leaf damage in Mimulus guttatus exhibits transgenerational plasticity mediated through differential expression of hundreds of genes. The epigenetic mechanisms that facilitate this response have yet to be described. Results We performed whole genome bisulfite sequencing in the progeny of genetically identical damaged and control plants and developed a pipeline to compare differences in the mean and variance of methylation between treatment groups. We find that parental damage increases the variability of CG and CHG methylation among progeny, but does not alter the overall mean methylation. Instead it has positive effects in some regions and negative in others. We find 3,396 CHH, 203 CG, and 54 CHG Differentially Methylated Regions (DMRs) ranging from tens to thousands of base pairs scattered across the genome. CHG and CHH DMRs tended to overlap with transposable elements. CG DMRs tended to overlap with gene coding regions, many of which were previously found to be differentially expressed. Conclusions Genome-wide increases in methylome variation suggest that parental conditions can increase epigenetic diversity in response to stress. Additionally, the potential association between CG DMRs and differentially expressed genes supports the hypothesis that differential methylation is a mechanistic component of transgenerational plasticity in M. guttatus.University of Kansas Botany EndowmentNSF IOS-0951254Kansas-INBRE P20-GM10341

DNA methylation and gene expression in Mimulus guttatus

Background The presence of methyl groups on cytosine nucleotides across an organism’s genome (methylation) is a major regulator of genome stability, crossing over, and gene regulation. The capacity for DNA methylation to be altered by environmental conditions, and potentially passed between generations, makes it a prime candidate for transgenerational epigenetic inheritance. Here we conduct the first analysis of the Mimulus guttatus methylome, with a focus on the relationship between DNA methylation and gene expression. Results We present a whole genome methylome for the inbred line Iron Mountain 62 (IM62). DNA methylation varies across chromosomes, genomic regions, and genes. We develop a model that predicts gene expression based on DNA methylation (R2 = 0.2). Post hoc analysis of this model confirms prior relationships, and identifies novel relationships between methylation and gene expression. Additionally, we find that DNA methylation is significantly depleted near gene transcriptional start sites, which may explain the recently discovered elevated rate of recombination in these same regions. Conclusions The establishment here of a reference methylome will be a useful resource for the continued advancement of M. guttatus as a model system. Using a model-based approach, we demonstrate that methylation patterns are an important predictor of variation in gene expression. This model provides a novel approach for differential methylation analysis that generates distinct and testable hypotheses regarding gene expression

Predicting evolutionary change at the DNA level in a natural Mimulus population

Evolution by natural selection occurs when the frequencies of genetic variants change because individuals differ in Darwinian fitness components such as survival or reproductive success. Differential fitness has been demonstrated in field studies of many organisms, but it remains unclear how well we can quantitatively predict allele frequency changes from fitness measurements. Here, we characterize natural selection on millions of Single Nucleotide Polymorphisms (SNPs) across the genome of the annual plant Mimulus guttatus. We use fitness estimates to calibrate population genetic models that effectively predict allele frequency changes into the next generation. Hundreds of SNPs experienced “male selection” in 2013 with one allele at each SNP elevated in frequency among successful male gametes relative to the entire population of adults. In the following generation, allele frequencies at these SNPs consistently shifted in the predicted direction. A second year of study revealed that SNPs had effects on both viability and reproductive success with pervasive trade-offs between fitness components. SNPs favored by male selection were, on average, detrimental to survival. These trade-offs (antagonistic pleiotropy and temporal fluctuations in fitness) may be essential to the long-term maintenance of alleles. Despite the challenges of measuring selection in the wild, the strong correlation between predicted and observed allele frequency changes suggests that population genetic models have a much greater role to play in forward-time prediction of evolutionary change

piRNAs Are Associated with Diverse Transgenerational Effects on Gene and Transposon Expression in a Hybrid Dysgenic Syndrome of D. virilis

Sexual reproduction allows transposable elements (TEs) to proliferate, leading to rapid divergence between populations and species. A significant outcome of divergence in the TE landscape is evident in hybrid dysgenic syndromes, a strong form of genomic incompatibility that can arise when (TE) family abundance differs between two parents. When TEs inherited from the father are absent in the mother's genome, TEs can become activated in the progeny, causing germline damage and sterility. Studies in Drosophila indicate that dysgenesis can occur when TEs inherited paternally are not matched with a pool of corresponding TE silencing PIWI-interacting RNAs (piRNAs) provisioned by the female germline. Using the D. virilis syndrome of hybrid dysgenesis as a model, we characterize the effects that divergence in TE profile between parents has on offspring. Overall, we show that divergence in the TE landscape is associated with persisting differences in germline TE expression when comparing genetically identical females of reciprocal crosses and these differences are transmitted to the next generation. Moreover, chronic and persisting TE expression coincides with increased levels of genic piRNAs associated with reduced gene expression. Combined with these effects, we further demonstrate that gene expression is idiosyncratically influenced by differences in the genic piRNA profile of the parents that arise though polymorphic TE insertions. Overall, these results support a model in which early germline events in dysgenesis establish a chronic, stable state of both TE and gene expression in the germline that is maintained through adulthood and transmitted to the next generation. This work demonstrates that divergence in the TE profile is associated with diverse piRNA-mediated transgenerational effects on gene expression within populations

Data from: Transgenerational effects alter plant defense and resistance in nature

Trichomes, or leaf hairs, are epidermal extensions that take a variety of forms and perform many functions in plants, including herbivore defense. In this study, I document genetically determined variation, within-generation plasticity, and a direct role of trichomes in herbivore defense for (Mimulus guttatus). After establishing the relationship between trichomes and herbivory, I test for transgenerational effects of wounding on trichome density and herbivore resistance. Patterns of inter-annual variation in herbivore density and the high cost of plant defense makes plant-herbivore interactions a system in which transgenerational phenotypic plasticity (TPP) is apt to evolve. Here, I demonstrate that parental damage alters offspring trichome density and herbivore resistance in nature. Moreover, this response varies between populations. This is among the first studies to demonstrate that TPP contributes to variation in nature, and also suggests that selection can modify TPP in response to local conditions

JC_JEB_Trichome_Herbivory

Three sheets containing trichome and herbivory data from field studies, tests of within generation plasticity, and transgenerational effects respectively

Mimulus sRNAs Are Wound Responsive and Associated with Transgenerationally Plastic Genes but Rarely Both

Organisms alter development in response to environmental cues. Recent studies demonstrate that they can transmit this plasticity to progeny. While the phenotypic and transcriptomic evidence for this “transgenerational plasticity” has accumulated, genetic and developmental mechanisms remain unclear. Plant defenses, gene expression and DNA methylation are modified as an outcome of parental wounding in Mimulus guttatus. Here, we sequenced M. guttatus small RNAs (sRNA) to test their possible role in mediating transgenerational plasticity. We sequenced sRNA populations of leaf-wounded and control plants at 1 h and 72 h after damage and from progeny of wounded and control parents. This allowed us to test three components of an a priori model of sRNA mediated transgenerational plasticity—(1) A subset of sRNAs will be differentially expressed in response to wounding, (2) these will be associated with previously identified differentially expressed genes and differentially methylated regions and (3) changes in sRNA abundance in wounded plants will be predictive of sRNA abundance, DNA methylation, and/or gene expression shifts in the following generation. Supporting (1) and (2), we found significantly different sRNA abundances in wounded leaves; the majority were associated with tRNA fragments (tRFs) rather than small-interfering RNAs (siRNA). However, siRNAs responding to leaf wounding point to Jasmonic Acid mediated responses in this system. We found that different sRNA classes were associated with regions of the genome previously found to be differentially expressed or methylated in progeny of wounded plants. Evidence for (3) was mixed. We found that non-dicer sRNAs with increased abundance in response to wounding tended to be nearby genes with decreased expression in the next generation. Counter to expectations, we did not find that siRNA responses to wounding were associated with gene expression or methylation changes in the next generation and within plant and transgenerational sRNA plasticity were negatively correlated

Parental experience modifies the Mimulus methylome

Abstract Background Transgenerational plasticity occurs when the environmental experience of an organism modifies the growth and development of its progeny. Leaf damage in Mimulus guttatus exhibits transgenerational plasticity mediated through differential expression of hundreds of genes. The epigenetic mechanisms that facilitate this response have yet to be described. Results We performed whole genome bisulfite sequencing in the progeny of genetically identical damaged and control plants and developed a pipeline to compare differences in the mean and variance of methylation between treatment groups. We find that parental damage increases the variability of CG and CHG methylation among progeny, but does not alter the overall mean methylation. Instead it has positive effects in some regions and negative in others. We find 3,396 CHH, 203 CG, and 54 CHG Differentially Methylated Regions (DMRs) ranging from tens to thousands of base pairs scattered across the genome. CHG and CHH DMRs tended to overlap with transposable elements. CG DMRs tended to overlap with gene coding regions, many of which were previously found to be differentially expressed. Conclusions Genome-wide increases in methylome variation suggest that parental conditions can increase epigenetic diversity in response to stress. Additionally, the potential association between CG DMRs and differentially expressed genes supports the hypothesis that differential methylation is a mechanistic component of transgenerational plasticity in M. guttatus