Whole-Genome DNA Methylation Analysis in Brassica rapa subsp. perviridis in Response to Albugo candida Infection.

DNA methylation is an epigenetic mark associated with several mechanisms in plants including immunity mechanisms. However, little is known about the regulatory role of DNA methylation in the resistance response of Brassica species against fungal diseases. White rust, caused by the fungus Albugo candida, is one of the most widespread and destructive diseases of all the cultivated Brassica species, particularly Brassica rapa L. and Brassica juncea (L.) Czern and Coss. Here, we investigate whole-genome DNA methylation modifications of B. rapa subsp. perviridis in response to white rust. As a result, 233 and 275 differentially methylated regions (DMRs) in the susceptible cultivar "Misugi" and the resistant cultivar "Nanane" were identified, respectively. In both cultivars, more than half of the DMRs were associated with genes (DMR-genes). Gene expression analysis showed that 13 of these genes were also differentially expressed between control and infected samples. Gene ontology enrichment analysis of DMR genes revealed their involvement in various biological processes including defense mechanisms. DMRs were unevenly distributed around genes in susceptible and resistant cultivars. In "Misugi," DMRs tended to be located within genes, while in "Nanane," DMRs tended to be located up and downstream of the genes. However, CG DMRs were predominantly located within genes in both cultivars. Transposable elements also showed association with all three sequence contexts of DMRs but predominantly with CHG and CHH DMRs in both cultivars. Our findings indicate the occurrence of DNA methylation modifications in B. rapa in response to white rust infection and suggest a potential regulatory role of DNA methylation modification in defense mechanisms which could be exploited to improve disease resistance.

Transcriptional regulation of genes bearing intronic heterochromatin in the rice genome.

Intronic regions of eukaryotic genomes accumulate many Transposable Elements (TEs). Intronic TEs often trigger the formation of transcriptionally repressive heterochromatin, even within transcription-permissive chromatin environments. Although TE-bearing introns are widely observed in eukaryotic genomes, their epigenetic states, impacts on gene regulation and function, and their contributions to genetic diversity and evolution, remain poorly understood. In this study, we investigated the genome-wide distribution of intronic TEs and their epigenetic states in the Oryza sativa genome, where TEs comprise 35% of the genome. We found that over 10% of rice genes contain intronic heterochromatin, most of which are associated with TEs and repetitive sequences. These heterochromatic introns are longer and highly enriched in promoter-proximal positions. On the other hand, introns also accumulate hypomethylated short TEs. Genes with heterochromatic introns are implicated in various biological functions. Transcription of genes bearing intronic heterochromatin is regulated by an epigenetic mechanism involving the conserved factor OsIBM2, mutation of which results in severe developmental and reproductive defects. Furthermore, we found that heterochromatic introns evolve rapidly compared to non-heterochromatic introns. Our study demonstrates that heterochromatin is a common epigenetic feature associated with actively transcribed genes in the rice genome.

Patterns of genomic differentiation between two Lake Victoria cichlid species, Haplochromis pyrrhocephalus and H. sp. 'macula'.

BACKGROUND: The molecular basis of the incipient stage of speciation is still poorly understood. Cichlid fish species in Lake Victoria are a prime example of recent speciation events and a suitable system to study the adaptation and reproductive isolation of species. RESULTS: Here, we report the pattern of genomic differentiation between two Lake Victoria cichlid species collected in sympatry, Haplochromis pyrrhocephalus and H. sp. 'macula,' based on the pooled genome sequences of 20 individuals of each species. Despite their ecological differences, population genomics analyses demonstrate that the two species are very close to a single panmictic population due to extensive gene flow. However, we identified 21 highly differentiated short genomic regions with fixed nucleotide differences. At least 15 of these regions contained genes with predicted roles in adaptation and reproductive isolation, such as visual adaptation, circadian clock, developmental processes, adaptation to hypoxia, and sexual selection. The nonsynonymous fixed differences in one of these genes, LWS, were reported as substitutions causing shift in absorption spectra of LWS pigments. Fixed differences were found in the promoter regions of four other differentially expressed genes, indicating that these substitutions may alter gene expression levels. CONCLUSIONS: These diverged short genomic regions may have contributed to the differentiation of two ecologically different species. Moreover, the origins of adaptive variants within the differentiated regions predate the geological formation of Lake Victoria; thus Lake Victoria cichlid species diversified via selection on standing genetic variation.

Single-cell expression noise and gene-body methylation in Arabidopsis thaliana.

Gene-body methylation (gbM) refers to an increased level of methylated cytosines specifically in a CG sequence context within genes. gbM is found in plant genes with intermediate expression level, which evolve slowly, and is often broadly conserved across millions of years of evolution. Intriguingly however, some plants lack gbM, and thus it remains unclear whether gbM has a function. In animals, there is support for a role of gbM in reducing erroneous transcription and transcription noise, but so far most studies in plants have tested for an effect of gbM on expression level, not noise. Here, we therefore tested whether gbM was associated with reduced expression noise in Arabidopsis thaliana, using single-cell transcriptome sequencing data from root quiescent centre cells. We find that gbM genes have lower expression noise levels than unmethylated genes. However, an analysis of covariance revealed that, if other genomic features are taken into account, this association disappears. Nonetheless, gbM genes were more consistently expressed across single-cell samples, supporting previous inference that gbM genes are constitutively expressed. Finally, we observed that fewer RNAseq reads map to introns of gbM genes than to introns of unmethylated genes, which indicates that gbM might be involved in reducing erroneous transcription by reducing intron retention.

Genome-wide characterization of DNA methylation, small RNA expression, and histone H3 lysine nine di-methylation in Brassica rapa L.

Epigenetic gene regulation is crucial to plant life and can involve dynamic interactions between various histone modifications, DNA methylation, and small RNAs. Detailed analysis of epigenome information is anticipated to reveal how the DNA sequence of the genome is translated into the plant's phenotype. The aim of this study was to map the DNA methylation state at the whole genome level and to clarify the relationship between DNA methylation and transcription, small RNA expression, and histone H3 lysine 9 di-methylation (H3K9me2) in Brassica rapa. We performed whole genome bisulfite sequencing, small RNA sequencing, and chromatin immunoprecipitation sequencing using H3K9me2 antibody in a Chinese cabbage inbred line, RJKB-T24, and examined the impact of epigenetic states on transcription. Cytosine methylation in DNA was analysed in different sequence contexts (CG, CHG, and CHH) (where H could be A, C, or T) and position (promoter, exon, intron, terminator, interspersed repeat regions), and the H3K9me2 and 24 nucleotide small interfering RNAs (24 nt-siRNA) were overlaid onto the B. rapa reference genome. The epigenome was compared with that of Arabidopsis thaliana and the relationship between the position of DNA methylation and gene expression, and the involvement of 24 nt siRNAs and H3K9me2 are discussed.

Coalescent framework for prokaryotes undergoing interspecific homologous recombination.

Coalescent process for prokaryote species is theoretically considered. Prokaryotes undergo homologous recombination with individuals of the same species (intraspecific recombination) and with individuals of other species (interspecific recombination). This work particularly focuses on interspecific recombination because intraspecific recombination has been well incorporated in coalescent framework. We present a simulation framework for generating SNP (single-nucleotide polymorphism) patterns that allows external DNA integration into host genome from other species. Using this simulation tool, msPro, we observed that the joint processes of intra- and interspecific recombination generate complex SNP patterns. The direct effect of interspecific recombination includes increased polymorphism. Because interspecific recombination is very rare in nature, it generates regions with exceptionally high polymorphism. Following interspecific recombination, intraspecific recombination cuts the integrated external DNA into small fragments, generating a complex SNP pattern that appears as if external DNA was integrated multiple times. The insight gained from our work using the msPro simulator will be useful for understanding and evaluating the relative contributions of intra- and interspecific recombination events in generating complex SNP patters in prokaryotes.

The Evolutionary Dynamics of Orthologs That Shift in Gene Body Methylation between Arabidopsis Species.

DNA methylation labels a specific subset of genes in plant genomes. Recent work has shown that this gene-body methylation (gbM) is a conserved feature of orthologs, because highly methylated genes in one species tend to be highly methylated in another. In this study, we examined the exceptions to that rule by identifying genes that differ in gbM status between two plant species-Arabidopsis thaliana and Arabidopsis lyrata. Using Capsella grandiflora as an outgroup, we polarized the loss and gain of gbM for orthologs in the Arabidopsis lineage. Our survey identified a few hundred genes that differed between ingroup species, out of ∼9,000 orthologs. The estimated rate of gbM gain was ∼2 × 10-9 per gene per year for both ingroup taxa and was similar to the loss rate in A. lyrata. In contrast, A. thaliana had a ∼3-fold higher estimated rate of gbM loss per gene, consistent with a recent diminishment of genome size. As in previous studies, we found that body-methylated genes were expressed broadly across tissues and were also longer than other genic sets. Genes that differed in gbM status exhibited higher variance in expression between species than genes that were body-methylated in both species. Moreover, the gain of gbM in one lineage tended to be associated with an increase of expression in that lineage. The genes that varied in gbM status between species varied more significantly in length between species than other sets of genes; we hypothesize that length is a key feature in the transition between body-methylated and nonmethylated genes.

Evolutionary patterns of genic DNA methylation vary across land plants.

Little is known about patterns of genic DNA methylation across the plant kingdom or about the evolutionary processes that shape them. To characterize gene-body methylation (gbM) within exons, we have gathered single-base resolution methylome data that span the phylogenetic breadth of land plants. We find that a basal land plant, Marchantia polymorpha, lacks any evident signal of gbM within exons, but conifers have high levels of both CG and CHG (where H is A, C or T) methylation in expressed genes. To begin to understand the evolutionary forces that shape gbM, we first tested for correlations in methylation levels across orthologues(1,2). Genic CG methylation levels, but not CHG or CHH levels, are correlated across orthologues for species as distantly related as ferns and angiosperms. Hence, relative levels of CG methylation are a consistent property across genes, even for species that diverged ∼400 million years ago(3,4). In contrast, genic CHG methylation correlates with genome size, suggesting that the host epigenetic response to transposable elements also affects genes. Altogether, our data indicate that the evolutionary forces acting on DNA methylation vary substantially across species, genes and methylation contexts.

CG Methylation Covaries with Differential Gene Expression between Leaf and Floral Bud Tissues of Brachypodium distachyon.

DNA methylation has the potential to influence plant growth and development through its influence on gene expression. To date, however, the evidence from plant systems is mixed as to whether patterns of DNA methylation vary significantly among tissues and, if so, whether these differences affect tissue-specific gene expression. To address these questions, we analyzed both bisulfite sequence (BSseq) and transcriptomic sequence data from three biological replicates of two tissues (leaf and floral bud) from the model grass species Brachypodium distachyon. Our first goal was to determine whether tissues were more differentiated in DNA methylation than explained by variation among biological replicates. Tissues were more differentiated than biological replicates, but the analysis of replicated data revealed high (>50%) false positive rates for the inference of differentially methylated sites (DMSs) and differentially methylated regions (DMRs). Comparing methylation to gene expression, we found that differential CG methylation consistently covaried negatively with gene expression, regardless as to whether methylation was within genes, within their promoters or even within their closest transposable element. The relationship between gene expression and either CHG or CHH methylation was less consistent. In total, CG methylation in promoters explained 9% of the variation in tissue-specific expression across genes, suggesting that CG methylation is a minor but appreciable factor in tissue differentiation.

A role for palindromic structures in the cis-region of maize Sirevirus LTRs in transposable element evolution and host epigenetic response.

Transposable elements (TEs) proliferate within the genome of their host, which responds by silencing them epigenetically. Much is known about the mechanisms of silencing in plants, particularly the role of siRNAs in guiding DNA methylation. In contrast, little is known about siRNA targeting patterns along the length of TEs, yet this information may provide crucial insights into the dynamics between hosts and TEs. By focusing on 6456 carefully annotated, full-length Sirevirus LTR retrotransposons in maize, we show that their silencing associates with underlying characteristics of the TE sequence and also uncover three features of the host-TE interaction. First, siRNA mapping varies among families and among elements, but particularly along the length of elements. Within the cis-regulatory portion of the LTRs, a complex palindrome-rich region acts as a hotspot of both siRNA matching and sequence evolution. These patterns are consistent across leaf, tassel, and immature ear libraries, but particularly emphasized for floral tissues and 21- to 22-nt siRNAs. Second, this region has the ability to form hairpins, making it a potential template for the production of miRNA-like, hairpin-derived small RNAs. Third, Sireviruses are targeted by siRNAs as a decreasing function of their age, but the oldest elements remain highly targeted, partially by siRNAs that cross-map to the youngest elements. We show that the targeting of older Sireviruses reflects their conserved palindromes. Altogether, we hypothesize that the palindromes aid the silencing of active elements and influence transposition potential, siRNA targeting levels, and ultimately the fate of an element within the genome.

Independent Molecular Basis of Convergent Highland Adaptation in Maize.

Convergent evolution is the independent evolution of similar traits in different species or lineages of the same species; this often is a result of adaptation to similar environments, a process referred to as convergent adaptation. We investigate here the molecular basis of convergent adaptation in maize to highland climates in Mesoamerica and South America, using genome-wide SNP data. Taking advantage of archaeological data on the arrival of maize to the highlands, we infer demographic models for both populations, identifying evidence of a strong bottleneck and rapid expansion in South America. We use these models to then identify loci showing an excess of differentiation as a means of identifying putative targets of natural selection and compare our results to expectations from recently developed theory on convergent adaptation. Consistent with predictions across a wide parameter space, we see limited evidence for convergent evolution at the nucleotide level in spite of strong similarities in overall phenotypes. Instead, we show that selection appears to have predominantly acted on standing genetic variation and that introgression from wild teosinte populations appears to have played a role in highland adaptation in Mexican maize.

Epigenetic regulation of intragenic transposable elements impacts gene transcription in Arabidopsis thaliana.

Genomes of higher eukaryotes, including plants, contain numerous transposable elements (TEs), that are often silenced by epigenetic mechanisms, such as histone modifications and DNA methylation. Although TE silencing adversely affects expression of nearby genes, recent studies reveal the presence of intragenic TEs marked by repressive heterochromatic epigenetic marks within transcribed genes. However, even for the well-studied plant model Arabidopsis thaliana, the abundance of intragenic TEs, how they are epigenetically regulated, and their potential impacts on host gene expression, remain unexplored. In this study, we comprehensively analyzed genome-wide distribution and epigenetic regulation of intragenic TEs in A. thaliana. Our analysis revealed that about 3% of TEs are located within gene bodies, dominantly at intronic regions. Most of them are shorter and less methylated than intergenic TEs, but they are still targeted by RNA-directed DNA methylation-dependent and independent pathways. Surprisingly, the heterochromatic epigenetic marks at TEs are maintained within actively transcribed genes. Moreover, the heterochromatic state of intronic TEs is critical for proper transcription of associated genes. Our study provides the first insight into how intragenic TEs affect the transcriptional landscape of the A. thaliana genome, and suggests the importance of epigenetic mechanisms for regulation of TEs within transcriptional gene units.

Population genomics of the fission yeast Schizosaccharomyces pombe.

The fission yeast Schizosaccharomyces pombe has been widely used as a model eukaryote to study a diverse range of biological processes. However, population genetic studies of this species have been limited to date, and we know very little about the evolutionary processes and selective pressures that are shaping its genome. Here, we sequenced the genomes of 32 worldwide S. pombe strains and examined the pattern of polymorphisms across their genomes. In addition to introns and untranslated regions (UTRs), intergenic regions also exhibited lower levels of nucleotide diversity than synonymous sites, suggesting that a considerable amount of noncoding DNA is under selective constraint and thus likely to be functional. A number of genomic regions showed a reduction of nucleotide diversity probably caused by selective sweeps. We also identified a region close to the end of chromosome 3 where an extremely high level of divergence was observed between 5 of the 32 strains and the remain 27, possibly due to introgression, strong positive selection, or that region being responsible for reproductive isolation. Our study should serve as an important starting point in using a population genomics approach to further elucidate the biology of this important model organism.

QTL map meets population genomics: an application to rice.

Genes involved in the transition from wild to cultivated crop species should be of great agronomic importance. Population genomic approaches utilizing genome resequencing data have been recently applied for this purpose, although it only reports a large list of candidate genes with no biological information. Here, by resequencing more than 30 genomes altogether of wild rice Oryza rufipogon and cultivated rice O. sativa, we identified a number of regions with clear footprints of selection during the domestication process. We then focused on identifying candidate domestication genes in these regions by utilizing the wealth of QTL information in rice. We were able to identify a number of interesting candidates such as transcription factors that should control key domestication traits such as shattering, awn length, and seed dormancy. Other candidates include those that might have been related to the improvement of grain quality and those that might have been involved in the local adaptation to dry conditions and colder environments. Our study shows that population genomic approaches and QTL mapping information can be used together to identify genes that might be of agronomic importance.

Identical sets of methylated and nonmethylated genes in Ciona intestinalis sperm and muscle cells.

BACKGROUND: The discovery of gene body methylation, which refers to DNA methylation within gene coding region, suggests an as yet unknown role of DNA methylation at actively transcribed genes. In invertebrates, gene bodies are the primary targets of DNA methylation, and only a subset of expressed genes is modified. RESULTS: Here we investigate the tissue variability of both the global levels and distribution of 5-methylcytosine (5mC) in the sea squirt Ciona intestinalis. We find that global 5mC content of early developmental embryos is high, but is strikingly reduced in body wall tissues. We chose sperm and adult muscle cells, with high and reduced levels of global 5mC respectively, for genome-wide analysis of 5mC targets. By means of CXXC-affinity purification followed by deep sequencing (CAP-seq), and genome-wide bisulfite sequencing (BS-seq), we designated body-methylated and unmethylated genes in each tissue. Surprisingly, body-methylated and unmethylated gene groups are identical in the sperm and muscle cells. Our analysis of microarray expression data shows that gene body methylation is associated with broad expression throughout development. Moreover, transgenic analysis reveals contrasting gene body methylation at an identical gene-promoter combination when integrated at different genomic sites. CONCLUSIONS: We conclude that gene body methylation is not a direct regulator of tissue specific gene expression in C. intestinalis. Our findings reveal constant targeting of gene body methylation irrespective of cell type, and they emphasize a correlation between gene body methylation and ubiquitously expressed genes. Our transgenic experiments suggest that the promoter does not determine the methylation status of the associated gene body.

Comparative analyses identify the contributions of exotic donors to disease resistance in a barley experimental population.

Introgression of novel genetic variation into breeding populations is frequently required to facilitate response to new abiotic or biotic pressure. This is particularly true for the introduction of host pathogen resistance in plant breeding. However, the number and genomic location of loci contributed by donor parents are often unknown, complicating efforts to recover desired agronomic phenotypes. We examined allele frequency differentiation in an experimental barley breeding population subject to introgression and subsequent selection for Fusarium head blight resistance. Allele frequency differentiation between the experimental population and the base population identified three primary genomic regions putatively subject to selection for resistance. All three genomic regions have been previously identified by quantitative trait locus (QTL) and association mapping. Based on the degree of identity-by-state relative to donor parents, putative donors of resistance alleles were also identified. The successful application of comparative population genetic approaches in this barley breeding experiment suggests that the approach could be applied to other breeding populations that have undergone defined breeding and selection histories, with the potential to provide valuable information for genetic improvement.

From many, one: genetic control of prolificacy during maize domestication.

A reduction in number and an increase in size of inflorescences is a common aspect of plant domestication. When maize was domesticated from teosinte, the number and arrangement of ears changed dramatically. Teosinte has long lateral branches that bear multiple small ears at their nodes and tassels at their tips. Maize has much shorter lateral branches that are tipped by a single large ear with no additional ears at the branch nodes. To investigate the genetic basis of this difference in prolificacy (the number of ears on a plant), we performed a genome-wide QTL scan. A large effect QTL for prolificacy (prol1.1) was detected on the short arm of chromosome 1 in a location that has previously been shown to influence multiple domestication traits. We fine-mapped prol1.1 to a 2.7 kb "causative region" upstream of the grassy tillers1 (gt1) gene, which encodes a homeodomain leucine zipper transcription factor. Tissue in situ hybridizations reveal that the maize allele of prol1.1 is associated with up-regulation of gt1 expression in the nodal plexus. Given that maize does not initiate secondary ear buds, the expression of gt1 in the nodal plexus in maize may suppress their initiation. Population genetic analyses indicate positive selection on the maize allele of prol1.1, causing a partial sweep that fixed the maize allele throughout most of domesticated maize. This work shows how a subtle cis-regulatory change in tissue specific gene expression altered plant architecture in a way that improved the harvestability of maize.

The Capsella rubella genome and the genomic consequences of rapid mating system evolution.

The shift from outcrossing to selfing is common in flowering plants, but the genomic consequences and the speed at which they emerge remain poorly understood. An excellent model for understanding the evolution of self fertilization is provided by Capsella rubella, which became self compatible <200,000 years ago. We report a C. rubella reference genome sequence and compare RNA expression and polymorphism patterns between C. rubella and its outcrossing progenitor Capsella grandiflora. We found a clear shift in the expression of genes associated with flowering phenotypes, similar to that seen in Arabidopsis, in which self fertilization evolved about 1 million years ago. Comparisons of the two Capsella species showed evidence of rapid genome-wide relaxation of purifying selection in C. rubella without a concomitant change in transposable element abundance. Overall we document that the transition to selfing may be typified by parallel shifts in gene expression, along with a measurable reduction of purifying selection.

QTL-seq: rapid mapping of quantitative trait loci in rice by whole genome resequencing of DNA from two bulked populations.

The majority of agronomically important crop traits are quantitative, meaning that they are controlled by multiple genes each with a small effect (quantitative trait loci, QTLs). Mapping and isolation of QTLs is important for efficient crop breeding by marker-assisted selection (MAS) and for a better understanding of the molecular mechanisms underlying the traits. However, since it requires the development and selection of DNA markers for linkage analysis, QTL analysis has been time-consuming and labor-intensive. Here we report the rapid identification of plant QTLs by whole-genome resequencing of DNAs from two populations each composed of 20-50 individuals showing extreme opposite trait values for a given phenotype in a segregating progeny. We propose to name this approach QTL-seq as applied to plant species. We applied QTL-seq to rice recombinant inbred lines and F2 populations and successfully identified QTLs for important agronomic traits, such as partial resistance to the fungal rice blast disease and seedling vigor. Simulation study showed that QTL-seq is able to detect QTLs over wide ranges of experimental variables, and the method can be generally applied in population genomics studies to rapidly identify genomic regions that underwent artificial or natural selective sweeps.

Gene body methylation is conserved between plant orthologs and is of evolutionary consequence.

DNA methylation is a common feature of eukaryotic genomes and is especially common in noncoding regions of plants. Protein coding regions of plants are often methylated also, but the extent, function, and evolutionary consequences of gene body methylation remain unclear. Here we investigate gene body methylation using an explicit comparative evolutionary approach. We generated bisulfite sequencing data from two tissues of Brachypodium distachyon and compared genic methylation patterns to those of rice (Oryza sativa ssp. japonica). Gene body methylation was strongly conserved between orthologs of the two species and affected a biased subset of long, slowly evolving genes. Because gene body methylation is conserved over evolutionary time, it shapes important features of plant genome evolution, such as the bimodality of G+C content among grass genes. Our results superficially contradict previous observations of high cytosine methylation polymorphism within Arabidopsis thaliana genes, but reanalyses of these data are consistent with conservation of methylation within gene regions. Overall, our results indicate that the methylation level is a long-term property of individual genes and therefore of evolutionary consequence.