Genomic basis of seed colour in quinoa inferred from variant patterns using extreme gradient boosting.

Quinoa is an agriculturally important crop species originally domesticated in the Andes of central South America. One of its most important phenotypic traits is seed colour. Seed colour variation is determined by contrasting abundance of betalains, a class of strong antioxidant and free radicals scavenging colour pigments only found in plants of the order Caryophyllales. However, the genetic basis for these pigments in seeds remains to be identified. Here we demonstrate the application of machine learning (extreme gradient boosting) to identify genetic variants predictive of seed colour. We show that extreme gradient boosting outperforms the classical genome-wide association approach. We provide re-sequencing and phenotypic data for 156 South American quinoa accessions and identify candidate genes potentially controlling betalain content in quinoa seeds. Genes identified include novel cytochrome P450 genes and known members of the betalain synthesis pathway, as well as genes annotated as being involved in seed development. Our work showcases the power of modern machine learning methods to extract biologically meaningful information from large sequencing data sets.

Molecular signatures of alternative reproductive strategies in a facultatively social hover wasp.

Social insect reproductives and non-reproductives represent ideal models with which to understand the expression and regulation of alternative phenotypes. Most research in this area has focused on the developmental regulation of reproductive phenotypes in obligately social taxa such as honey bees, while relatively few studies have addressed the molecular correlates of reproductive differentiation in species in which the division of reproductive labour is established only in plastic dominance hierarchies. To address this knowledge gap, we generate the first genome for any stenogastrine wasp and analyse brain transcriptomic data for non-reproductives and reproductives of the facultatively social species Liostenogaster flavolineata, a representative of one of the simplest forms of social living. By experimentally manipulating the reproductive 'queues' exhibited by social colonies of this species, we show that reproductive division of labour in this species is associated with transcriptomic signatures that are more subtle and variable than those observed in social taxa in which colony living has become obligate; that variation in gene expression among non-reproductives reflects their investment into foraging effort more than their social rank; and that genes associated with reproductive division of labour overlap to some extent with those underlying division of labour in the separate polistine origin of wasp sociality but only explain a small portion of overall variation in this trait. These results indicate that broad patterns of within-colony transcriptomic differentiation in this species are similar to those in Polistinae but offer little support for the existence of a strongly conserved 'toolkit' for sociality.

Prediction of NB-LRR resistance genes based on full-length sequence homology.

The activation of plant immunity is mediated by resistance (R)-gene receptors, also known as nucleotide-binding leucine-rich repeat (NB-LRR) genes, which in turn trigger the authentic defense response. R-gene identification is a crucial goal for both classic and modern plant breeding strategies for disease resistance. The conventional method identifies NB-LRR genes using a protein motif/domain-based search (PDS) within an automatically predicted gene set of the respective genome assembly. PDS proved to be imprecise since repeat masking prior to automatic genome annotation unwittingly prevented comprehensive NB-LRR gene detection. Furthermore, R-genes have diversified in a species-specific manner, so that NB-LRR gene identification cannot be universally standardized. Here, we present the full-length Homology-based R-gene Prediction (HRP) method for the comprehensive identification and annotation of a genome's R-gene repertoire. Our method has substantially addressed the complex genomic organization of tomato (Solanum lycopersicum) NB-LRR gene loci, proving to be more performant than the well-established RenSeq approach. HRP efficiency was also tested on three differently assembled and annotated Beta sp. genomes. Indeed, HRP identified up to 45% more full-length NB-LRR genes compared to previous approaches. HRP also turned out to be a more refined strategy for R-gene allele mining, testified by the identification of hitherto undiscovered Fom-2 homologs in five Cucurbita sp. genomes. In summary, our high-performance method for full-length NB-LRR gene discovery will propel the identification of novel R-genes towards development of improved cultivars.

Genome-environment associations along elevation gradients in two snowbed species of the North-Eastern Calcareous Alps.

BACKGROUND: Anthropogenic climate change leads to increasing temperatures and altered precipitation and snowmelt patterns, especially in alpine ecosystems. To understand species' responses to climate change, assessment of genetic structure and diversity is crucial as the basis for the evaluation of migration patterns, genetic adaptation potential as well as the identification of adaptive alleles. RESULTS: We studied genetic structure, diversity and genome-environment associations of two snowbed species endemic to the Eastern Alps with a large elevational range, Achillea clusiana Tausch and Campanula pulla L. Genotyping-by-sequencing was employed to assemble loci de novo, call variants and perform population genetic analyses. Populations of either species were distinguishable by mountain, and to some extent by elevation. We found evidence for gene flow between elevations. Results of genome-environment associations suggested similar selective pressures acting on both species, emanating mainly from precipitation and exposition rather than temperature. CONCLUSIONS: Given their genetic structure and amount of gene flow among populations the two study species are suitable to serve as a model for genetic monitoring of climate change adaptation along an elevation gradient. Consequences of climate change will predominantly manifest via changes in precipitation and, thus, duration of snow cover in the snowbeds and indirectly via shrub encroachment accompanied by increasing shading of snowbeds at lower range margins. Assembling genomes of the study species and studying larger sample sizes and time series will be necessary to functionally characterize and validate the herein identified genomic loci putatively involved in adaptive processes.

Subgenome evolution in allotetraploid plants.

Polyploidization is a well-known speciation and adaptation mechanism. Traces of former polyploidization events were discovered within many genomes, and especially in plants. Allopolyploidization by interspecific hybridization between two species is common. Among hybrid plants, many are domesticated species of agricultural interest and many of their genomes and of their presumptive parents have been sequenced. Hybrid genomes remain challenging to analyse because of the presence of multiple subgenomes. The genomes of hybrids often undergo rearrangement and degradation over time. Based on 10 hybrid plant genomes from six different genera, with hybridization dating from 10,000 to 5 million years ago, we assessed subgenome degradation, subgenomic intermixing and biased subgenome fractionation. The restructuring of hybrid genomes does not proceed proportionally with the age of the hybrid. The oldest hybrids in our data set display completely different fates: whereas the subgenomes of the tobacco plant Nicotiana benthamiana are in an advanced stage of degradation, the subgenomes of quinoa (Chenopodium quinoa) are exceptionally well conserved by structure and sequence. We observed statistically significant biased subgenome fractionation in seven out of 10 hybrids, which had different ages and subgenomic intermixing levels. Hence, we conclude that no correlation exists between biased fractionation and subgenome intermixing. Lastly, domestication may encourage or hinder subgenome intermixing, depending on the evolutionary context. In summary, comparative analysis of hybrid genomes and their presumptive parents allowed us to determine commonalities and differences between their evolutionary fates. In order to facilitate the future analysis of further hybrid genomes, we automated the analysis steps within manticore, which is publicly available at https://github.com/MatteoSchiavinato/manticore.git.

Parental origin of the allotetraploid tobacco Nicotiana benthamiana.

Nicotiana section Suaveolentes is an almost all-Australian clade of allopolyploid tobacco species including the important plant model Nicotiana benthamiana. The homology relationships of this clade and its formation are not completely understood. To address this gap, we assessed phylogenies of all individual genes of N. benthamiana and the well studied N. tabacum (section Nicotiana) and their homologues in six diploid Nicotiana species. We generated sets of 44 424 and 65 457 phylogenetic trees of N. benthamiana and N. tabacum genes, respectively, each collectively called a phylome. Members of Nicotiana sections Noctiflorae and Sylvestres were represented as the species closest to N. benthamiana in most of the gene trees. Analyzing the gene trees of the phylome we: (i) dated the hybridization event giving rise to N. benthamiana to 4-5 MyA, and (ii) separated the subgenomes. We assigned 1.42 Gbp of the genome sequence to section Noctiflorae and 0.97 Gbp to section Sylvestres based on phylome analysis. In contrast, read mapping of the donor species did not succeed in separating the subgenomes of N. benthamiana. We show that the maternal progenitor of N. benthamiana was a member of section Noctiflorae, and confirm a member of section Sylvestres as paternal subgenome donor. We also demonstrate that the advanced stage of long-term genome diploidization in N. benthamiana is reflected in its subgenome organization. Taken together, our results underscore the usefulness of phylome analysis for subgenome characterization in hybrid species.

Quinoa genome assembly employing genomic variation for guided scaffolding.

KEY MESSAGE: We propose to use the natural variation between individuals of a population for genome assembly scaffolding. In today's genome projects, multiple accessions get sequenced, leading to variant catalogs. Using such information to improve genome assemblies is attractive both cost-wise as well as scientifically, because the value of an assembly increases with its contiguity. We conclude that haplotype information is a valuable resource to group and order contigs toward the generation of pseudomolecules. Quinoa (Chenopodium quinoa) has been under cultivation in Latin America for more than 7500 years. Recently, quinoa has gained increasing attention due to its stress resistance and its nutritional value. We generated a novel quinoa genome assembly for the Bolivian accession CHEN125 using PacBio long-read sequencing data (assembly size 1.32 Gbp, initial N50 size 608 kbp). Next, we re-sequenced 50 quinoa accessions from Peru and Bolivia. This set of accessions differed at 4.4 million single-nucleotide variant (SNV) positions compared to CHEN125 (1.4 million SNV positions on average per accession). We show how to exploit variation in accessions that are distantly related to establish a genome-wide ordered set of contigs for guided scaffolding of a reference assembly. The method is based on detecting shared haplotypes and their expected continuity throughout the genome (i.e., the effect of linkage disequilibrium), as an extension of what is expected in mapping populations where only a few haplotypes are present. We test the approach using Arabidopsis thaliana data from different populations. After applying the method on our CHEN125 quinoa assembly we validated the results with mate-pairs, genetic markers, and another quinoa assembly originating from a Chilean cultivar. We show consistency between these information sources and the haplotype-based relations as determined by us and obtain an improved assembly with an N50 size of 1079 kbp and ordered contig groups of up to 39.7 Mbp. We conclude that haplotype information in distantly related individuals of the same species is a valuable resource to group and order contigs according to their adjacency in the genome toward the generation of pseudomolecules.

Genomes of the wild beets Beta patula and Beta vulgaris ssp. maritima.

We present draft genome assemblies of Beta patula, a critically endangered wild beet endemic to the Madeira archipelago, and of the closely related Beta vulgaris ssp. maritima (sea beet). Evidence-based reference gene sets for B. patula and sea beet were generated, consisting of 25 127 and 27 662 genes, respectively. The genomes and gene sets of the two wild beets were compared with their cultivated sister taxon B. vulgaris ssp. vulgaris (sugar beet). Large syntenic regions were identified, and a display tool for automatic genome-wide synteny image generation was developed. Phylogenetic analysis based on 9861 genes showing 1:1:1 orthology supported the close relationship of B. patula to sea beet and sugar beet. A comparative analysis of the Rz2 locus, responsible for rhizomania resistance, suggested that the sequenced B. patula accession was rhizomania susceptible. Reference karyotypes for the two wild beets were established, and genomic rearrangements were detected. We consider our data as highly valuable and comprehensive resources for wild beet studies, B. patula conservation management, and sugar beet breeding research.

Comparative genome characterization of the periodontal pathogen Tannerella forsythia.

BACKGROUND: Tannerella forsythia is a bacterial pathogen implicated in periodontal disease. Numerous virulence-associated T. forsythia genes have been described, however, it is necessary to expand the knowledge on T. forsythia's genome structure and genetic repertoire to further elucidate its role within pathogenesis. Tannerella sp. BU063, a putative periodontal health-associated sister taxon and closest known relative to T. forsythia is available for comparative analyses. In the past, strain confusion involving the T. forsythia reference type strain ATCC 43037 led to discrepancies between results obtained from in silico analyses and wet-lab experimentation. RESULTS: We generated a substantially improved genome assembly of T. forsythia ATCC 43037 covering 99% of the genome in three sequences. Using annotated genomes of ten Tannerella strains we established a soft core genome encompassing 2108 genes, based on orthologs present in > = 80% of the strains analysed. We used a set of known and hypothetical virulence factors for comparisons in pathogenic strains and the putative periodontal health-associated isolate Tannerella sp. BU063 to identify candidate genes promoting T. forsythia's pathogenesis. Searching for pathogenicity islands we detected 38 candidate regions in the T. forsythia genome. Only four of these regions corresponded to previously described pathogenicity islands. While the general protein O-glycosylation gene cluster of T. forsythia ATCC 43037 has been described previously, genes required for the initiation of glycan synthesis are yet to be discovered. We found six putative glycosylation loci which were only partially conserved in other bacteria. Lastly, we performed a comparative analysis of translational bias in T. forsythia and Tannerella sp. BU063 and detected highly biased genes. CONCLUSIONS: We provide resources and important information on the genomes of Tannerella strains. Comparative analyses enabled us to assess the suitability of T. forsythia virulence factors as therapeutic targets and to suggest novel putative virulence factors. Further, we report on gene loci that should be addressed in the context of elucidating T. forsythia's protein O-glycosylation pathway. In summary, our work paves the way for further molecular dissection of T. forsythia biology in general and virulence of this species in particular.

Container-aided integrative QTL and RNA-seq analysis of Collaborative Cross mice supports distinct sex-oriented molecular modes of response in obesity.

BACKGROUND: The Collaborative Cross (CC) mouse population is a valuable resource to study the genetic basis of complex traits, such as obesity. Although the development of obesity is influenced by environmental factors, underlying genetic mechanisms play a crucial role in the response to these factors. The interplay between the genetic background and the gene expression pattern can provide further insight into this response, but we lack robust and easily reproducible workflows to integrate genomic and transcriptomic information in the CC mouse population. RESULTS: We established an automated and reproducible integrative workflow to analyse complex traits in the CC mouse genetic reference panel at the genomic and transcriptomic levels. We implemented the analytical workflow to assess the underlying genetic mechanisms of host susceptibility to diet induced obesity and integrated these results with diet induced changes in the hepatic gene expression of susceptible and resistant mice. Hepatic gene expression differs significantly between obese and non-obese mice, with a significant sex effect, where male and female mice exhibit different responses and coping mechanisms. CONCLUSION: Integration of the data showed that different genes but similar pathways are involved in the genetic susceptibility and disturbed in diet induced obesity. Genetic mechanisms underlying susceptibility to high-fat diet induced obesity are different in female and male mice. The clear distinction we observed in the systemic response to the high-fat diet challenge and to obesity between male and female mice points to the need for further research into distinct sex-related mechanisms in metabolic disease.

nextPARS: parallel probing of RNA structures in Illumina.

RNA molecules play important roles in virtually every cellular process. These functions are often mediated through the adoption of specific structures that enable RNAs to interact with other molecules. Thus, determining the secondary structures of RNAs is central to understanding their function and evolution. In recent years several sequencing-based approaches have been developed that allow probing structural features of thousands of RNA molecules present in a sample. Here, we describe nextPARS, a novel Illumina-based implementation of in vitro parallel probing of RNA structures. Our approach achieves comparable accuracy to previous implementations, while enabling higher throughput and sample multiplexing.

The genome of the recently domesticated crop plant sugar beet (Beta vulgaris).

Sugar beet (Beta vulgaris ssp. vulgaris) is an important crop of temperate climates which provides nearly 30% of the world's annual sugar production and is a source for bioethanol and animal feed. The species belongs to the order of Caryophylalles, is diploid with 2n = 18 chromosomes, has an estimated genome size of 714-758 megabases and shares an ancient genome triplication with other eudicot plants. Leafy beets have been cultivated since Roman times, but sugar beet is one of the most recently domesticated crops. It arose in the late eighteenth century when lines accumulating sugar in the storage root were selected from crosses made with chard and fodder beet. Here we present a reference genome sequence for sugar beet as the first non-rosid, non-asterid eudicot genome, advancing comparative genomics and phylogenetic reconstructions. The genome sequence comprises 567 megabases, of which 85% could be assigned to chromosomes. The assembly covers a large proportion of the repetitive sequence content that was estimated to be 63%. We predicted 27,421 protein-coding genes supported by transcript data and annotated them on the basis of sequence homology. Phylogenetic analyses provided evidence for the separation of Caryophyllales before the split of asterids and rosids, and revealed lineage-specific gene family expansions and losses. We sequenced spinach (Spinacia oleracea), another Caryophyllales species, and validated features that separate this clade from rosids and asterids. Intraspecific genomic variation was analysed based on the genome sequences of sea beet (Beta vulgaris ssp. maritima; progenitor of all beet crops) and four additional sugar beet accessions. We identified seven million variant positions in the reference genome, and also large regions of low variability, indicating artificial selection. The sugar beet genome sequence enables the identification of genes affecting agronomically relevant traits, supports molecular breeding and maximizes the plant's potential in energy biotechnology.

Genome and transcriptome characterization of the glycoengineered Nicotiana benthamiana line ΔXT/FT.

BACKGROUND: The allotetraploid tobacco species Nicotiana benthamiana native to Australia has become a popular host for recombinant protein production. Although its usage grows every year, little is known on this plant's genomic and transcriptomic features. Most N. benthamiana accessions currently used in research lack proper documentation of their breeding history and provenance. One of these, the glycoengineered N. benthamiana line ΔXT/FT is increasingly used for the production of biopharmaceutical proteins. RESULTS: Based on an existing draft assembly of the N. benthamiana genome we predict 50,516 protein -encoding genes (62,216 transcripts) supported by expression data derived from 2.35 billion mRNA-seq reads. Using single-copy core genes we show high completeness of the predicted gene set. We functionally annotate more than two thirds of the gene set through sequence homology to genes from other Nicotiana species. We demonstrate that the expression profiles from leaf tissue of ΔXT/FT and its wild type progenitor only show minimal differences. We identify the transgene insertion sites in ΔXT/FT and show that one of the transgenes was inserted inside another predicted gene that most likely lost its function upon insertion. Based on publicly available mRNA-seq data, we confirm that the N. benthamiana accessions used by different research institutions most likely derive from a single source. CONCLUSIONS: This work provides gene annotation of the N. benthamiana genome, a genomic and transcriptomic characterization of a transgenic N. benthamiana line in comparison to its wild-type progenitor, and sheds light onto the relatedness of N. benthamiana accessions that are used in laboratories around the world.

Architecture and evolution of a minute plant genome.

It has been argued that the evolution of plant genome size is principally unidirectional and increasing owing to the varied action of whole-genome duplications (WGDs) and mobile element proliferation. However, extreme genome size reductions have been reported in the angiosperm family tree. Here we report the sequence of the 82-megabase genome of the carnivorous bladderwort plant Utricularia gibba. Despite its tiny size, the U. gibba genome accommodates a typical number of genes for a plant, with the main difference from other plant genomes arising from a drastic reduction in non-genic DNA. Unexpectedly, we identified at least three rounds of WGD in U. gibba since common ancestry with tomato (Solanum) and grape (Vitis). The compressed architecture of the U. gibba genome indicates that a small fraction of intergenic DNA, with few or no active retrotransposons, is sufficient to regulate and integrate all the processes required for the development and reproduction of a complex organism.

Oligoadenylation of 3' decay intermediates promotes cytoplasmic mRNA degradation in Drosophila cells.

Post-transcriptional 3' end addition of nucleotides is important in a variety of RNA decay pathways. We have examined the 3' end addition of nucleotides during the decay of the Hsp70 mRNA and a corresponding reporter RNA in Drosophila S2 cells by conventional sequencing of cDNAs obtained after mRNA circularization and by deep sequencing of dedicated libraries enriched for 3' decay intermediates along the length of the mRNA. Approximately 5%-10% of 3' decay intermediates carried nonencoded oligo(A) tails with a mean length of 2-3 nucleotides. RNAi experiments showed that the oligoadenylated RNA fragments were intermediates of exosomal decay and the noncanonical poly(A) polymerase Trf4-1 was mainly responsible for A addition. A hot spot of A addition corresponded to an intermediate of 3' decay that accumulated upon inhibition of decapping, and knockdown of Trf4-1 increased the abundance of this intermediate, suggesting that oligoadenylation facilitates 3' decay. Oligoadenylated 3' decay intermediates were found in the cytoplasmic fraction in association with ribosomes, and fluorescence microscopy revealed a cytoplasmic localization of Trf4-1. Thus, oligoadenylation enhances exosomal mRNA degradation in the cytoplasm.

Molecular signatures of plastic phenotypes in two eusocial insect species with simple societies.

Phenotypic plasticity is important in adaptation and shapes the evolution of organisms. However, we understand little about what aspects of the genome are important in facilitating plasticity. Eusocial insect societies produce plastic phenotypes from the same genome, as reproductives (queens) and nonreproductives (workers). The greatest plasticity is found in the simple eusocial insect societies in which individuals retain the ability to switch between reproductive and nonreproductive phenotypes as adults. We lack comprehensive data on the molecular basis of plastic phenotypes. Here, we sequenced genomes, microRNAs (miRNAs), and multiple transcriptomes and methylomes from individual brains in a wasp (Polistes canadensis) and an ant (Dinoponera quadriceps) that live in simple eusocial societies. In both species, we found few differences between phenotypes at the transcriptional level, with little functional specialization, and no evidence that phenotype-specific gene expression is driven by DNA methylation or miRNAs. Instead, phenotypic differentiation was defined more subtly by nonrandom transcriptional network organization, with roles in these networks for both conserved and taxon-restricted genes. The general lack of highly methylated regions or methylome patterning in both species may be an important mechanism for achieving plasticity among phenotypes during adulthood. These findings define previously unidentified hypotheses on the genomic processes that facilitate plasticity and suggest that the molecular hallmarks of social behavior are likely to differ with the level of social complexity.

Diversification, evolution and methylation of short interspersed nuclear element families in sugar beet and related Amaranthaceae species.

Short interspersed nuclear elements (SINEs) are non-autonomous non-long terminal repeat retrotransposons which are widely distributed in eukaryotic organisms. While SINEs have been intensively studied in animals, only limited information is available about plant SINEs. We analysed 22 SINE families from seven genomes of the Amaranthaceae family and identified 34 806 SINEs, including 19 549 full-length copies. With the focus on sugar beet (Beta vulgaris), we performed a comparative analysis of the diversity, genomic and chromosomal organization and the methylation of SINEs to provide a detailed insight into the evolution and age of Amaranthaceae SINEs. The lengths of consensus sequences of SINEs range from 113 nucleotides (nt) up to 224 nt. The SINEs show dispersed distribution on all chromosomes but were found with higher incidence in subterminal euchromatic chromosome regions. The methylation of SINEs is increased compared with their flanking regions, and the strongest effect is visible for cytosines in the CHH context, indicating an involvement of asymmetric methylation in the silencing of SINEs.

Whole-genome sequencing identifies recurrent mutations in chronic lymphocytic leukaemia.

Chronic lymphocytic leukaemia (CLL), the most frequent leukaemia in adults in Western countries, is a heterogeneous disease with variable clinical presentation and evolution. Two major molecular subtypes can be distinguished, characterized respectively by a high or low number of somatic hypermutations in the variable region of immunoglobulin genes. The molecular changes leading to the pathogenesis of the disease are still poorly understood. Here we performed whole-genome sequencing of four cases of CLL and identified 46 somatic mutations that potentially affect gene function. Further analysis of these mutations in 363 patients with CLL identified four genes that are recurrently mutated: notch 1 (NOTCH1), exportin 1 (XPO1), myeloid differentiation primary response gene 88 (MYD88) and kelch-like 6 (KLHL6). Mutations in MYD88 and KLHL6 are predominant in cases of CLL with mutated immunoglobulin genes, whereas NOTCH1 and XPO1 mutations are mainly detected in patients with unmutated immunoglobulins. The patterns of somatic mutation, supported by functional and clinical analyses, strongly indicate that the recurrent NOTCH1, MYD88 and XPO1 mutations are oncogenic changes that contribute to the clinical evolution of the disease. To our knowledge, this is the first comprehensive analysis of CLL combining whole-genome sequencing with clinical characteristics and clinical outcomes. It highlights the usefulness of this approach for the identification of clinically relevant mutations in cancer.

Profiling of extensively diversified plant LINEs reveals distinct plant-specific subclades.

A large fraction of eukaryotic genomes is made up of long interspersed nuclear elements (LINEs). Due to their capability to create novel copies via error-prone reverse transcription, they generate multiple families and reach high copy numbers. Although mammalian LINEs have been well described, plant LINEs have been only poorly investigated. Here, we present a systematic cross-species survey of LINEs in higher plant genomes shedding light on plant LINE evolution as well as diversity, and facilitating their annotation in genome projects. Applying a Hidden Markov Model (HMM)-based analysis, 59 390 intact LINE reverse transcriptases (RTs) were extracted from 23 plant genomes. These fall in only two out of 28 LINE clades (L1 and RTE) known in eukaryotes. While plant RTE LINEs are highly homogenous and mostly constitute only a single family per genome, plant L1 LINEs are extremely diverse and form numerous families. Despite their heterogeneity, all members across the 23 species fall into only seven L1 subclades, some of them defined here. Exemplarily focusing on the L1 LINEs of a basal reference plant genome (Beta vulgaris), we show that the subclade classification level does not only reflect RT sequence similarity, but also mirrors structural aspects of complete LINE retrotransposons, like element size, position and type of encoded enzymatic domains. Our comprehensive catalogue of plant LINE RTs serves the classification of highly diverse plant LINEs, while the provided subclade-specific HMMs facilitate their annotation.

The CHH motif in sugar beet satellite DNA: a modulator for cytosine methylation.

Methylation of DNA is important for the epigenetic silencing of repetitive DNA in plant genomes. Knowledge about the cytosine methylation status of satellite DNAs, a major class of repetitive DNA, is scarce. One reason for this is that arrays of tandemly arranged sequences are usually collapsed in next-generation sequencing assemblies. We applied strategies to overcome this limitation and quantified the level of cytosine methylation and its pattern in three satellite families of sugar beet (Beta vulgaris) which differ in their abundance, chromosomal localization and monomer size. We visualized methylation levels along pachytene chromosomes with respect to small satellite loci at maximum resolution using chromosome-wide fluorescent in situ hybridization complemented with immunostaining and super-resolution microscopy. Only reduced methylation of many satellite arrays was obtained. To investigate methylation at the nucleotide level we performed bisulfite sequencing of 1569 satellite sequences. We found that the level of methylation of cytosine strongly depends on the sequence context: cytosines in the CHH motif show lower methylation (44-52%), while CG and CHG motifs are more strongly methylated. This affects the overall methylation of satellite sequences because CHH occurs frequently while CG and CHG are rare or even absent in the satellite arrays investigated. Evidently, CHH is the major target for modulation of the cytosine methylation level of adjacent monomers within individual arrays and contributes to their epigenetic function. This strongly indicates that asymmetric cytosine methylation plays a role in the epigenetic modification of satellite repeats in plant genomes.