In plants, CG DNA methylation is prevalent in the transcribed regions of many constitutively expressed genes (gene body methylation; gbM), but the origin and function of gbM remain unknown. Here we report the discovery that Eutrema salsugineum has lost gbM from its genome, to our knowledge the first instance for an angiosperm. Of all known DNA methyltransferases, only CHROMOMETHYLASE 3 (CMT3) is missing from E. salsugineum. Identification of an additional angiosperm, Conringia planisiliqua, which independently lost CMT3 and gbM, supports that CMT3 is required for the establishment of gbM. Detailed analyses of gene expression, the histone variant H2A.Z, and various histone modifications in E. salsugineum and in Arabidopsis thaliana epigenetic recombinant inbred lines found no evidence in support of any role for gbM in regulating transcription or affecting the composition and modification of chromatin over evolutionary timescales.DNA methylation | gene body methylation | epigenetics | histone modifications | CHROMOMETHYLASE 3 I n angiosperms, cytosine DNA methylation occurs in three sequence contexts: Methylated CG (mCG) is catalyzed by METHYLTRANSFERASE 1 (MET1), mCHG (where H is A/C/T) by CHROMOMETHYLASE 3 (CMT3), and mCHH by DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2) or CHROMOMETHYLASE 2 (CMT2) (1). MET1 performs a maintenance function and is targeted by VARIANT IN METHYLATION 1 (VIM1), which binds preexisting hemimethylated CG sites. In contrast, DRM2 is targeted by RNA-directed DNA methylation (RdDM) for the de novo establishment of mCHH. CMT3 forms a self-reinforcing loop with the H3K9me2 pathway to maintain mCHG; however, considering that transformation of CMT3 into the cmt3 background can rescue DNA methylation defects, it is reasonable to also consider CMT3 a de novo methyltransferase (2). Two main lines of evidence suggest that DNA methylation plays an important role in the transcriptional silencing of transposable elements (TEs): that TEs are usually methylated, and that the loss of DNA methylation (e.g., in methyltransferase mutants) is often accompanied by TE reactivation.A large number of plant genes (e.g., ∼13.5% of all Arabidopsis thaliana genes) also contain exclusively mCG in the transcribed region and a depletion of mCG from both the transcriptional start and stop sites (referred to as "gene body DNA methylation"; gbM) ( Fig. 1A) (3)(4)(5). A survey of plant methylome data showed that the emergence of gbM in the plant kingdom is specific to angiosperms (6), whereas nonflowering plants (such as mosses and green algae) have much more diverse genic methylation patterns (7,8). Similar to mCG at TEs, the maintenance of gbM requires MET1. In contrast to DNA methylation at TEs, however, gbM does not appear to be associated with transcriptional repression. Rather, genes containing gbM are ubiquitously expressed at moderate to high levels compared with non-gbM genes (4, 5, 9), and within gbM genes there is a correlation between transcript abundance and methylation levels (10, 11).It has been proposed ...

Resequencing or reference-based assemblies reveal large parts of the small-scale sequence variation. However, they typically fail to separate such local variation into colinear and rearranged variation, because they usually do not recover the complement of large-scale rearrangements, including transpositions and inversions. Besides the availability of hundreds of genomes of diverse Arabidopsis thaliana accessions, there is so far only one full-length assembled genome: the reference sequence. We have assembled 117 Mb of the A. thaliana Landsberg erecta (Ler) genome into five chromosome-equivalent sequences using a combination of short Illumina reads, long PacBio reads, and linkage information. Whole-genome comparison against the reference sequence revealed 564 transpositions and 47 inversions comprising ∼3.6 Mb, in addition to 4.1 Mb of nonreference sequence, mostly originating from duplications. Although rearranged regions are not different in local divergence from colinear regions, they are drastically depleted for meiotic recombination in heterozygotes. Using a 1.2-Mb inversion as an example, we show that such rearrangement-mediated reduction of meiotic recombination can lead to genetically isolated haplotypes in the worldwide population of A. thaliana. Moreover, we found 105 single-copy genes, which were only present in the reference sequence or the Ler assembly, and 334 single-copy orthologs, which showed an additional copy in only one of the genomes. To our knowledge, this work gives first insights into the degree and type of variation, which will be revealed once complete assemblies will replace resequencing or other reference-dependent methods.

Genes underlying mutant phenotypes can be isolated by combining marker discovery, genetic mapping and resequencing, but a more straightforward strategy for mapping mutations would be the direct comparison of mutant and wild-type genomes. Applying such an approach, however, is hampered by the need for reference sequences and by mutational loads that confound the unambiguous identification of causal mutations. Here we introduce NIKS (needle in the k-stack), a reference-free algorithm based on comparing k-mers in whole-genome sequencing data for precise discovery of homozygous mutations. We applied NIKS to eight mutants induced in nonreference rice cultivars and to two mutants of the nonmodel species Arabis alpina. In both species, comparing pooled F 2 individuals selected for mutant phenotypes revealed small sets of mutations including the causal changes. Moreover, comparing M 3 seedlings of two allelic mutants unambiguously identified the causal gene. Thus, for any species amenable to mutagenesis, NIKS enables forward genetics without requiring segregating populations, genetic maps and reference sequences.Forward genetic screens have been of fundamental importance in elucidating biological mechanisms in model species 1 . Their success, however, has relied on the feasibility of mutant gene isolation. Identification of causal mutations typically begins with genetic mapping, followed by candidate gene sequencing and complementation studies using transformation. Advances in DNA sequencing technologies have tremendously accelerated genetic mapping by combining bulk segregant analysis, that is, pooling recombinant genomes, with whole-genome sequencing, usually referred to as mapping by sequencing 2,3 . This approach is now becoming standard for mutation mapping and identification in many model species 3-12 and has even been applied to decipher quantitative traits with complex genetic architectures 13,14 . Recently, mutagen-induced changes have been used as novel markers, allowing mapping of mutations using isogenic mapping populations 10,15 . Nevertheless, all mapping-by-sequencing methods rely on resequencing, a method for whole-genome reconstruction based on aligning sequences to a reference sequence. Therefore, this requirement restricts the application of the technique to species for which such a reference genome sequence is available.Many reference-sequence assembly projects are currently in progress, including ones for most of the major crop species and breeding animals. However, even with an existing reference sequence, extending mapping-by-sequencing methods beyond the sequenced reference accessions has proved technically challenging. Mutant alleles of genes that are not present in the reference sequence cannot be identified within resequencing data alone. In particular, fast-evolving genes, such as those involved in disease resistance, might not always be represented in the reference sequence 16,17 .Alternative solutions for mapping-by-sequencing in species without reference sequences have been proposed, such ...

Mapping-by-sequencing (or SHOREmapping) has revitalized the powerful concept of forward genetic screens in plants. However, as in conventional genetic mapping approaches, mapping-by-sequencing requires phenotyping of mapping populations established from crosses between two diverged accessions. In addition to the segregation of the focal phenotype, this introduces natural phenotypic variation, which can interfere with the recognition of quantitative phenotypes. Here, we demonstrate how mapping-by-sequencing and candidate gene identification can be performed within the same genetic background using only mutagen-induced changes as segregating markers. Using a previously unknown suppressor of mutants of like heterochromatin protein1 (lhp1), which in its functional form is involved in chromatin-mediated gene repression, we identified three closely linked ethyl methanesulfonate-induced changes as putative candidates. In order to assess allele frequency differences between such closely linked mutations, we introduced deep candidate resequencing using the new Ion Torrent Personal Genome Machine sequencing platform to our mutant identification pipeline and thereby reduced the number of causal candidate mutations to only one. Genetic analysis of two independent additional alleles confirmed that this mutation was causal for the suppression of lhp1.