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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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Tirnaz, Miyaji, Takuno, Bayer, Shimizu, Akter, Edwards, Batley and Fujimoto.)

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., Competing Interests: The authors have declared that no competing interests exist.

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.

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.

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 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.

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., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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.

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., (© 2016 Bousios et al.; Published by Cold Spring Harbor Laboratory Press.)

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., (Copyright © 2015 by the Genetics Society of America.)

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., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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.

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.

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.

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.

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.

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., Competing Interests: The authors have declared that no competing interests exist.

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., (© 2013 The Authors The Plant Journal © 2013 Blackwell Publishing Ltd.)

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.

Gene duplication plays a crucial role in the development of complex biosystems, but the evolutionary forces behind the growth of biosystems are poorly understood. In this work, we introduce a model for such a growth through gene duplication. Plant microRNAs (miRNAs) are considered as a model. miRNAs are one of the non-coding small RNAs (19-25 nucleotides), which are involved in the post-transcriptional gene regulation. A single kind of miRNAs can be encoded by multiple genomic regions called miRNA genes, and can regulate multiple kinds of functional gene families. It is assumed that a single miRNA system involves all these genes, miRNA genes and their target gene families. We are interested in how duplication of miRNA genes affects the evolution of the miRNA system by focusing on the numbers of miRNA genes and their target gene families, denoted by x and y, respectively. We here theoretically explore the evolutionary growth of (x,y); the former increases by duplication of the miRNA gene while the latter increases when an independent gene family acquires a novel binding site of the miRNA by mutations. We first investigate the evolutionary patterns of (x,y) under three commonly assumed scenarios for the evolution of duplicated genes, that is, the positive and negative dosage and neofunctionalization scenarios. The results indicate that under the three scenarios, the transient process of (x,y) is unidirectional, although the direction is different depending on the model. This pattern is not consistent with the observation in the Arabidopsis thaliana genome, suggesting that a model that incorporates at least two directional evolutionary forces is needed to explain the observation. Then, such a model called the "complexity growth model" is introduced, in which we assume that duplication of miRNA genes is evolutionary advantageous in that the system can encode a complex and sophisticated pattern of regulation because multiple miRNA genes can have different expression patterns. This is helpful to optimize the regulation of a few particular functional gene families, but there is a cost; once the system is optimized for one purpose, it could be difficult for other purposes to use it. That is, duplication of miRNA genes would narrow down the potential gene families that can join the system. Our theoretical analysis revealed that this model can explain the observation of Arabidopsis miRNAs. Although we consider plant miRNAs as an example in this work, the model can be readily applied to other regulation systems with some modifications. Further development of such models would provide insights into the evolutionary growth of the complexity of biosystems., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

In plants, the genome of the host responds to the amplification of transposable elements (TEs) with DNA methylation. However, neither the factors involved in TE methylation nor the dynamics of the host-TE interaction are well resolved. Here, we identify 5,522 long terminal repeat retrotransposons (LTR-RT) in the genome of Oryza sativa ssp. japonica and then assess methylation for individual elements. Our analyses uncover three strong trends: long LTR-RTs are more highly methylated, the insertion times of LTR-RTs are negatively correlated with methylation, and young LTR-RTs tend to be closer to genes than older insertions. Additionally, a phylogenetic examination of the gypsy-like LTR-RT superfamily revealed that methylation is phylogenetically correlated. Given these observations, we present a model suggesting that the phylogenetic correlation among related LTR-RTs is a primary mechanism driving methylation. In this model, bursts of transposition produce new elements with high sequence similarity. The host machinery identifies proliferating elements as well as closely related LTR-RTs through cross-homology. In addition, our data are consistent with previous hypotheses that methylated LTR-RT elements are removed preferentially from regions near genes, explaining some of the observed age distribution.

We analyzed the genome-wide pattern of single nucleotide polymorphisms (SNPs) in a sample with 12 strains of Staphylococcus aureus. Population structure of S. aureus seems to be complex, and the 12 strains were divided into five groups, named A, B, C, D, and E. We conducted a detailed analysis of the topologies of gene genealogies across the genomes and observed a high rate and frequency of tree-shape switching, indicating extensive homologous recombination. Most of the detected recombination occurred in the ancestral population of A, B, and C, whereas there are a number of small regions that exhibit evidence for homologous recombination with a distinct related species. As such regions would contain a number of novel mutations, it is suggested that homologous recombination would play a crucial role to maintain genetic variation within species. In the A-B-C ancestral population, we found multiple lines of evidence that the coalescent pattern is very similar to what is expected in a panmictic population, suggesting that this population is suitable to apply the standard population genetic theories. Our analysis showed that homologous recombination caused a dramatic decay in linkage disequilibrium (LD) and there is almost no LD between SNPs with distance more than 10 kb. Coalescent simulations demonstrated that a high rate of homologous recombination-a relative rate of 0.6 to the mutation rate with an average tract length of about 10 kb-is required to produce patterns similar to those observed in the S. aureus genomes. Our results call for more research into the evolutionary role of homologous recombination in bacterial populations.

DNA methylation of coding regions, known as gene body methylation, is conserved across eukaryotic lineages. The function of body methylation is not known, but it may either prevent aberrant expression from intragenic promoters or enhance the accuracy of splicing. Given these putative functions, we hypothesized that body-methylated genes would be both longer and more functionally important than unmethylated genes. To test these hypotheses, we reanalyzed single-base resolution bisulfite sequence data from Arabidopsis thaliana to differentiate body-methylated genes from unmethylated genes using a probabilistic approach. Contrasting genic characteristics between the two groups, we found that body-methylated genes tend to be longer and to be more functionally important, as measured by phenotypic effects of insertional mutants and by gene expression, than unmethylated genes. We also found that methylated genes evolve more slowly than unmethylated genes, despite the potential for increased mutation rates in methylated CpG dinucleotides. We propose that slower rates in body-methylated genes are a function of higher selective constraint, lower nucleosome occupancy, and a lower proportion of CpG dinucleotides.

QTL (quantitative trait loci) mapping is commonly used to identify genetic regions responsible to important phenotype variation. A common strategy of QTL mapping is to use recombinant inbred lines (RILs), which are usually established by several generations of inbreeding of an F1 population (usually up to F6 or F7 populations). As this inbreeding process involves a large amount of labor, we are particularly interested in the effect of the number of inbreeding generations on the power of QTL mapping; a part of the labor could be saved if a smaller number of inbreeding provides sufficient power. By using simulations, we investigated the performance of QTL mapping with recombinant inbred lines (RILs). As expected, we found that the power of F4 population could be almost comparable to that of F6 and F7 populations. A potential problem in using F4 population is that a large proportion of RILs are heterozygotes. We here introduced a new method to partly relax this problem. The performance of this method was verified by simulations with a wide range of parameters including the size of the segregation population, recombination rate, genome size and the density of markers. We found our method works better than the commonly used standard method especially when there are a number of heterozygous markers. Our results imply that in most cases, QTL mapping does not necessarily require RILs at F6 or F7 generations; rather, F4 (or even F3) populations would be almost as useful as F6 or F7 populations. Because the cost to establish a number of RILs for many generations is enormous, this finding will cause a reduction in the cost of QTL mapping, thereby accelerating gene mapping in many species.

MicroRNAs (miRNAs) are involved in posttranscriptional gene regulation by repressing the expression of their target genes through inhibition of translation and/or cleavage of mRNAs. In plants, the target genes of miRNAs usually belong to large gene families, and miRNA regulation is gained for individual genes throughout gene family evolution. To explore the selective effect of miRNA regulation on their target genes, we investigated the pattern of polymorphism and interspecific divergence in 11 multigene families that include target genes of ancient miRNAs in Arabidopsis thaliana. We found that the levels of polymorphism and divergence in target genes are significantly reduced, whereas those for nontarget genes are very similar to the genomic averages. This pattern is particularly clear at synonymous sites; we found that the reduction of synonymous variation is caused by selection for optimal codons, which increase the efficiency and accuracy of translation. Based on these results, we conclude that tuning via miRNA regulation has a strong impact on the evolution of target genes through which highly sophisticated regulation systems have been established.

The semidwarf phenotype has been extensively selected during modern crop breeding as an agronomically important trait. Introduction of the semidwarf gene, semi-dwarf1 (sd1), which encodes a gibberellin biosynthesis enzyme, made significant contributions to the "green revolution" in rice (Oryza sativa L.). Here we report that SD1 was involved not only in modern breeding including the green revolution, but also in early steps of rice domestication. We identified two SNPs in O. sativa subspecies (ssp.) japonica SD1 as functional nucleotide polymorphisms (FNPs) responsible for shorter culm length and low gibberellin biosynthetic activity. Genetic diversity analysis among O. sativa ssp. japonica and indica, along with their wild ancestor O. rufipogon Griff, revealed that these FNPs clearly differentiate the japonica landrace and O. rufipogon. We also found a dramatic reduction in nucleotide diversity around SD1 only in the japonica landrace, not in the indica landrace or O. rufipogon. These findings indicate that SD1 has been subjected to artificial selection in rice evolution and that the FNPs participated in japonica domestication, suggesting that ancient humans already used the green revolution gene.

Pseudogenes are defined as nonfunctional DNA sequences with homology to functional protein-coding genes, and they typically contain nonfunctional mutations within the presumptive coding region. In theory, pseudogenes can also be caused by mutations in upstream regulatory regions, appearing as open reading frames with attenuated expression. In this study, we identified 1,939 annotated protein-coding genes with little evidence of expression in Arabidopsis thaliana and characterized their molecular evolutionary characteristics. On average, this set of genes was shorter than expressed genes and evolved with a 2-fold higher rate of nonsynonymous substitutions. The divergence of upstream sequences, based on ortholog comparisons to A. lyrata, was also higher than expressed genes, suggesting that these lowly expressed genes could be examples of pseudogenization by promoter disablement, often due to transposable element insertion. We complemented our empirical study by extending the models of Force et al. (Force A, Lynch M, Pickett FB, Amores A, Yan YL, Postlethwait J. 1999. Preservation of duplicate genes by complementary, degenerative mutations. Genetics 151:1531-1545.) to derive the probability of promoter disablements after gene duplication.

Interlocus gene conversion is considered a crucial mechanism for generating novel combinations of polymorphisms in duplicated genes. The importance of gene conversion between duplicated genes has been recognized in the major histocompatibility complex and self-incompatibility genes, which are likely subject to diversifying selection. To theoretically understand the potential role of gene conversion in such situations, forward simulations are performed in various two-locus models. The results show that gene conversion could significantly increase the number of haplotypes when diversifying selection works on both loci. We find that the tract length of gene conversion is an important factor to determine the efficacy of gene conversion: shorter tract lengths can more effectively generate novel haplotypes given the gene conversion rate per site is the same. Similar results are also obtained when one of the duplicated genes is assumed to be a pseudogene. It is suggested that a duplicated gene, even after being silenced, will contribute to increasing the variability in the other locus through gene conversion. Consequently, the fixation probability and longevity of duplicated genes increase under the presence of gene conversion. On the basis of these findings, we propose a new scenario for the preservation of a duplicated gene: when the original donor gene is under diversifying selection, a duplicated copy can be preserved by gene conversion even after it is pseudogenized.

One of the causes of genome size expansion is considered to be amplification of retrotransposons. We determined nucleotide sequences of 24 PCR products for each of six retrotransposons in Brassica rapa and Brassica oleracea. Phylogenetic trees of these sequences showed species-specific clades. We also sequenced STF7a homologs and Tto1 homologs, 24 PCR products each, in nine diploids and three allopolyploids, and constructed phylogenetic trees. In these phylogenetic trees, species-specific clades of diploid species were also formed, but retrotransposons of allopolyploids were clustered into the clades of their original genomes, indicating that these two retrotransposons amplified after speciation of the nine diploids. Genetic variation in these retrotransposons may have arisen before emergence of allopolyploid species. There was a positive correlation between the genome size and the average number of substitutions of STF7a and Tto1 homologs in at least seven diploids. The implications of these results in the genome evolution of Brassicaceae are herein discussed.

Using Arabidopsis microRNA (miRNA)-mediated gene regulation system as a model, we investigated how complex systems evolve with special attention to selection to maintain the systems. We found that the copy number of miRNA genes within each system is a key factor to determine the complexity of the system, indicating a crucial role of gene duplication to increase the complexity. Furthermore, we show that the mode of selection to maintain the systems depend on their complexity levels.

In self-incompatibility, a number of S haplotypes are maintained by frequency-dependent selection, which results in trans-specific S haplotypes. The region of several kilobases (approximately 40-60 kb) from SP6 to SP2, including self-incompatibility-related genes and some adjacent genes in Brassica rapa, has high nucleotide diversity due to the hitchhiking effect, and therefore we call this region the "S-locus complex." Recombination in the S-locus complex is considered to be suppressed. We sequenced regions of >50 kb of the S-locus complex of three S haplotypes in B. rapa and found higher nucleotide diversity in intergenic regions than in coding regions. Two highly similar regions of >10 kb were found between BrS-8 and BrS-46. Phylogenetic analysis using trans-specific S haplotypes (called interspecific pairs) of B. rapa and B. oleracea suggested that recombination reduced the nucleotide diversity in these two regions and that the genes not involved in self-incompatibility in the S-locus complex and the kinase domain, but not the S domain, of SRK have also experienced recombination. Recombination may reduce hitchhiking diversity in the S-locus complex, whereas the region from the S domain to SP11 would disfavor recombination.