Danelle K. Seymour

Evolution of DNA Methylation Patterns in the Brassicaceae is Driven by Differences in Genome Organization

DNA methylation is an ancient molecular modification found in most eukaryotes. In plants, DNA methylation is not only critical for transcriptionally silencing transposons, but can also affect phenotype by altering expression of protein coding genes. The extent of its contribution to phenotypic diversity over evolutionary time is, however, unclear, because of limited stability of epialleles that are not linked to DNA mutations. To dissect the relative contribution of DNA methylation to transposon surveillance and host gene regulation, we leveraged information from three species in the Brassicaceae that vary in genome architecture, Capsella rubella, Arabidopsis lyrata, and Arabidopsis thaliana. We found that the lineage-specific expansion and contraction of transposon and repeat sequences is the main driver of interspecific differences in DNA methylation. The most heavily methylated portions of the genome are thus not conserved at the sequence level. Outside of repeat-associated methylation, there is a surprising degree of conservation in methylation at single nucleotides located in gene bodies. Finally, dynamic DNA methylation is affected more by tissue type than by environmental differences in all species, but these responses are not conserved. The majority of DNA methylation variation between species resides in hypervariable genomic regions, and thus, in the context of macroevolution, is of limited phenotypic consequence.

LNK genes integrate light and clock signaling networks at the core of the Arabidopsis oscillator

Light signaling pathways and the circadian clock interact to help organisms synchronize physiological and developmental processes with periodic environmental cycles. The plant photoreceptors responsible for clock resetting have been characterized, but signaling components that link the photoreceptors to the clock remain to be identified. Here we describe a family of night light–inducible and clock-regulated genes (LNK) that play a key role linking light regulation of gene expression to the control of daily and seasonal rhythms in Arabidopsis thaliana. A genomewide transcriptome analysis revealed that most light-induced genes respond more strongly to light during the subjective day, which is consistent with the diurnal nature of most physiological processes in plants. However, a handful of genes, including the homologous genes LNK1 and LNK2, are more strongly induced by light in the middle of the night, when the clock is most responsive to this signal. Further analysis revealed that the morning phased LNK1 and LNK2 genes control circadian rhythms, photomorphogenic responses, and photoperiodic dependent flowering, most likely by regulating a subset of clock and flowering time genes in the afternoon. LNK1 and LNK2 themselves are directly repressed by members of the TIMING OF CAB1 EXPRESSION/PSEUDO RESPONSE REGULATOR family of core-clock genes in the afternoon and early night. Thus, LNK1 and LNK2 integrate early light signals with temporal information provided by core oscillator components to control the expression of afternoon genes, allowing plants to keep track of seasonal changes in day length.

Rapid creation of Arabidopsis doubled haploid lines for quantitative trait locus mapping

Quantitative trait loci (QTL) mapping is a powerful tool for investigating the genetic basis of natural variation. QTL can be mapped using a number of different population designs, but recombinant inbred lines (RILs) are among the most effective. Unfortunately, homozygous RIL populations are time consuming to construct, typically requiring at least six generations of selfing starting from a heterozygous F1. Haploid plants produced from an F1 combine the two parental genomes and have only one allele at every locus. Converting these sterile haploids into fertile diploids (termed “doubled haploids,” DHs) produces immortal homozygous lines in only two steps. Here we describe a unique technique for rapidly creating recombinant doubled haploid populations in Arabidopsis thaliana: centromere-mediated genome elimination. We generated a population of 238 doubled haploid lines that combine two parental genomes and genotyped them by reduced representation Illumina sequencing. The recombination rate and parental allele frequencies in our population are similar to those found in existing RIL sets. We phenotyped this population for traits related to flowering time and for petiole length and successfully mapped QTL controlling each trait. Our work demonstrates that doubled haploid populations offer a rapid, easy alternative to RILs for Arabidopsis genetic analysis.

KH domain protein RCF3 is a tissue-biased regulator of the plant miRNA biogenesis cofactor HYL1

Significance Micro RNAs (miRNAs) are small RNA molecules that regulate gene expression posttranscriptionally in a process known as gene silencing. Fine-tuning the production of miRNAs is essential for correct silencing of their targets, which in turn is important for homeostasis and development. To fine-tune the production of miRNAs, plants deploy a combination of proteins that act as cofactors of the miRNA-processing machinery. Here, we describe REGULATOR OF CBF GENE EXPRESSION 3 (RCF3) as a tissue-specific regulator of miRNA biogenesis in plants. RCF3 interacts with the phosphatases C-TERMINAL DOMAIN PHOSPHATASE-LIKE1 and 2 (CPL1 and CPL2), ultimately affecting the phosphorylation of one of the main DICER-LIKE1 (DCL1) accessory proteins, HYPONASTIC LEAVES1 (HYL1), with a concomitant effect on miRNA production. The biogenesis of microRNAs (miRNAs), which regulate mRNA abundance through posttranscriptional silencing, comprises multiple well-orchestrated processing steps. We have identified the Arabidopsis thaliana K homology (KH) domain protein REGULATOR OF CBF GENE EXPRESSION 3 (RCF3) as a cofactor affecting miRNA biogenesis in specific plant tissues. MiRNA and miRNA-target levels were reduced in apex-enriched samples of rcf3 mutants, but not in other tissues. Mechanistically, RCF3 affects miRNA biogenesis through nuclear interactions with the phosphatases C-TERMINAL DOMAIN PHOSPHATASE-LIKE1 and 2 (CPL1 and CPL2). These interactions are essential to regulate the phosphorylation status, and thus the activity, of the double-stranded RNA binding protein and DICER-LIKE1 (DCL1) cofactor HYPONASTIC LEAVES1 (HYL1).

The causes and consequences of DNA methylome variation in plants.

Epigenetic variation - polymorphisms at the level of DNA methylation or histone modifications - modulates chromatin accessibility, which can perturb transcriptional activity and spur phenotypic variation. Determining the origin, frequency spectrum, and consequences of epigenetic variants is key to understanding the role of this variation in generating stable phenotypic variation in plants. Here we review recent literature on DNA methylation variation in both model and crop plant species with a focus on the link between genotype, epigenotype, and transcription. We highlight population epigenomics studies that explore the relationship between epigenetic variants and genetic diversity. Moreover, we provide an overview of relevant studies that together advocate a minor, albeit significant role for epigenetic variation in directing specific transcriptional changes.

Improving the Annotation of Arabidopsis lyrata Using RNA-Seq Data

Gene model annotations are important community resources that ensure comparability and reproducibility of analyses and are typically the first step for functional annotation of genomic regions. Without up-to-date genome annotations, genome sequences cannot be used to maximum advantage. It is therefore essential to regularly update gene annotations by integrating the latest information to guarantee that reference annotations can remain a common basis for various types of analyses. Here, we report an improvement of the Arabidopsis lyrata gene annotation using extensive RNA-seq data. This new annotation consists of 31,132 protein coding gene models in addition to 2,089 genes with high similarity to transposable elements. Overall, ~87% of the gene models are corroborated by evidence of expression and 2,235 of these models feature multiple transcripts. Our updated gene annotation corrects hundreds of incorrectly split or merged gene models in the original annotation, and as a result the identification of alternative splicing events and differential isoform usage are vastly improved.

Genetic architecture of nonadditive inheritance in Arabidopsis thaliana hybrids

Significance Hybrid progeny of inbred parents are often more fit than their parents. Such hybrid vigor, or heterosis, is the focus of many plant breeding programs, and the rewards are evident. Hybrid maize has for many decades accounted for the majority of seed planted each year in North America and Europe. Despite the prevalence of this phenomenon and its agricultural importance, the genetic basis of heterotic traits is still unclear. We have used a large collection of first-generation hybrids in Arabidopsis thaliana to characterize the genetics of heterosis in this model plant. We have identified loci that contribute substantially to hybrid vigor and show that a subset of these exhibits classical dominance, an important finding with direct implications for crop improvement. The ubiquity of nonparental hybrid phenotypes, such as hybrid vigor and hybrid inferiority, has interested biologists for over a century and is of considerable agricultural importance. Although examples of both phenomena have been subject to intense investigation, no general model for the molecular basis of nonadditive genetic variance has emerged, and prediction of hybrid phenotypes from parental information continues to be a challenge. Here we explore the genetics of hybrid phenotype in 435 Arabidopsis thaliana individuals derived from intercrosses of 30 parents in a half diallel mating scheme. We find that nonadditive genetic effects are a major component of genetic variation in this population and that the genetic basis of hybrid phenotype can be mapped using genome-wide association (GWA) techniques. Significant loci together can explain as much as 20% of phenotypic variation in the surveyed population and include examples that have both classical dominant and overdominant effects. One candidate region inherited dominantly in the half diallel contains the gene for the MADS-box transcription factor AGAMOUS-LIKE 50 (AGL50), which we show directly to alter flowering time in the predicted manner. Our study not only illustrates the promise of GWA approaches to dissect the genetic architecture underpinning hybrid performance but also demonstrates the contribution of classical dominance to genetic variance.

Methods for Genotyping-by-Sequencing

A major goal for biologists is to understand the connection between genes and phenotypic traits, and genetic mapping in experimental populations remains a powerful approach for discovering the causal genes underlying phenotypes. For genetic mapping, the process of genotyping was previously a major rate-limiting step. Modern sequencing technology has greatly improved the resolution and speed of genetic mapping by reducing the time, labor, and cost per genotyping marker. In addition, the ability to perform genotyping-by-sequencing (GBS) has facilitated large-scale population genetic analyses by providing a simpler way to survey segregating genetic variation in natural populations. Here we present two protocols for GBS, using the Illumina platform, that can be applied to a wide range of genotyping projects in different species. The first protocol is for genotyping a subset of marker positions genome-wide using restriction digestion, and the second is for preparing inexpensive paired-end whole-genome libraries. We discuss the suitability of each approach for different genotyping applications and provide notes for adapting these protocols for use with a liquid-handling robot.

Demography and its effects on genomic variation in crop domestication

Over two thousand plant species have been modified morphologically through cultivation and human use. Here, we review three aspects of crop domestication that are currently undergoing marked revisions, due to analytical advancements and their application to whole genome resequencing (WGS) data. We begin by discussing the duration and demographic history of domestication. There has been debate as to whether domestication occurred quickly or slowly. The latter is tentatively supported both by fossil data and application of WGS data to sequentially Markovian coalescent methods that infer the history of effective population size. This history suggests the possibility of extended human impacts on domesticated lineages prior to their purposeful cultivation. We also make the point that demographic history matters, because it shapes patterns and levels of extant genetic diversity. We illustrate this point by discussing the evolutionary processes that contribute to the empirical observation that most crops examined to date have more putatively deleterious alleles than their wild relatives. These deleterious alleles may contribute to genetic load within crops and may be fitting targets for crop improvement. Finally, the same demographic factors are likely to shape the spectrum of structural variants (SVs) within crops. SVs are known to underlie many of the phenotypic changes associated with domestication and crop improvement, but we currently lack sufficient knowledge about the mechanisms that create SVs, their rates of origin, their population frequencies and their phenotypic effects.A Review summarizes up-to-date understandings of the duration and demographic history of crop domestication and its effects on extant genomic variations, including structural variations about which we currently lack sufficient knowledge.

The genetic architecture of recurrent segregation distortion in Arabidopsis thaliana

The equal probability of transmission of alleles from either parent during sexual reproduction is a central tenet of genetics and evolutionary biology. Yet, there are many cases where this rule is violated. Such violations limit intraspecific gene flow and can facilitate the formation of genetic barriers, a first step in speciation. Biased transmission of alleles, or segregation distortion, can result from a number of biological processes including epistatic interactions between incompatible loci, gametic selection, and meiotic drive. Examples of these phenomena have been identified in many species, implying that they are universal, but comprehensive species-wide studies of segregation distortion are lacking. We have performed a species-wide screen for distorted allele frequencies in over 500 segregating populations of Arabidopsis thaliana using reduced-representation genome sequencing. Biased transmission of alleles was evident in up to a quarter of surveyed populations. Most populations exhibited distortion at only one genomic region, with some regions being repeatedly affected in multiple populations. Our results begin to elucidate the species-level architecture of biased transmission of genetic material in A. thaliana, and serve as a springboard for future studies into the basis of intraspecific genetic barriers.