Identification of a non-canonical ciliate nuclear genetic code where UAA and UAG code for different amino acids.

The genetic code is one of the most highly conserved features across life. Only a few lineages have deviated from the "universal" genetic code. Amongst the few variants of the genetic code reported to date, the codons UAA and UAG virtually always have the same translation, suggesting that their evolution is coupled. Here, we report the genome and transcriptome sequencing of a novel uncultured ciliate, belonging to the Oligohymenophorea class, where the translation of the UAA and UAG stop codons have changed to specify different amino acids. Genomic and transcriptomic analyses revealed that UAA has been reassigned to encode lysine, while UAG has been reassigned to encode glutamic acid. We identified multiple suppressor tRNA genes with anticodons complementary to the reassigned codons. We show that the retained UGA stop codon is enriched in the 3'UTR immediately downstream of the coding region of genes, suggesting that there is functional drive to maintain tandem stop codons. Using a phylogenomics approach, we reconstructed the ciliate phylogeny and mapped genetic code changes, highlighting the remarkable number of independent genetic code changes within the Ciliophora group of protists. According to our knowledge, this is the first report of a genetic code variant where UAA and UAG encode different amino acids.

Genome sequence and genetic diversity of European ash trees.

Ash trees (genus Fraxinus, family Oleaceae) are widespread throughout the Northern Hemisphere, but are being devastated in Europe by the fungus Hymenoscyphus fraxineus, causing ash dieback, and in North America by the herbivorous beetle Agrilus planipennis. Here we sequence the genome of a low-heterozygosity Fraxinus excelsior tree from Gloucestershire, UK, annotating 38,852 protein-coding genes of which 25% appear ash specific when compared with the genomes of ten other plant species. Analyses of paralogous genes suggest a whole-genome duplication shared with olive (Olea europaea, Oleaceae). We also re-sequence 37 F. excelsior trees from Europe, finding evidence for apparent long-term decline in effective population size. Using our reference sequence, we re-analyse association transcriptomic data, yielding improved markers for reduced susceptibility to ash dieback. Surveys of these markers in British populations suggest that reduced susceptibility to ash dieback may be more widespread in Great Britain than in Denmark. We also present evidence that susceptibility of trees to H. fraxineus is associated with their iridoid glycoside levels. This rapid, integrated, multidisciplinary research response to an emerging health threat in a non-model organism opens the way for mitigation of the epidemic.

An aphid RNA transcript migrates systemically within plants and is a virulence factor.

Aphids are sap-feeding insects that colonize a broad range of plant species and often cause feeding damage and transmit plant pathogens, including bacteria, viruses, and viroids. These insects feed from the plant vascular tissue, predominantly the phloem. However, it remains largely unknown how aphids, and other sap-feeding insects, establish intimate long-term interactions with plants. To identify aphid virulence factors, we took advantage of the ability of the green peach aphid Myzus persicae to colonize divergent plant species. We found that a M. persicae clone of near-identical females established stable colonies on nine plant species of five representative plant eudicot and monocot families that span the angiosperm phylogeny. Members of the novel aphid gene family Ya are differentially expressed in aphids on the nine plant species and are coregulated and organized as tandem repeats in aphid genomes. Aphids translocate Ya transcripts into plants, and some transcripts migrate to distal leaves within several plant species. RNAi-mediated knockdown of Ya genes reduces M. persicae fecundity, and M. persicae produces more progeny on transgenic plants that heterologously produce one of the systemically migrating Ya transcripts as a long noncoding (lnc) RNA. Taken together, our findings show that beyond a range of pathogens, M. persicae aphids translocate their own transcripts into plants, including a Ya lncRNA that migrates to distal locations within plants, promotes aphid fecundity, and is a member of a previously undescribed host-responsive aphid gene family that operate as virulence factors.

An improved assembly and annotation of the allohexaploid wheat genome identifies complete families of agronomic genes and provides genomic evidence for chromosomal translocations.

Advances in genome sequencing and assembly technologies are generating many high-quality genome sequences, but assemblies of large, repeat-rich polyploid genomes, such as that of bread wheat, remain fragmented and incomplete. We have generated a new wheat whole-genome shotgun sequence assembly using a combination of optimized data types and an assembly algorithm designed to deal with large and complex genomes. The new assembly represents >78% of the genome with a scaffold N50 of 88.8 kb that has a high fidelity to the input data. Our new annotation combines strand-specific Illumina RNA-seq and Pacific Biosciences (PacBio) full-length cDNAs to identify 104,091 high-confidence protein-coding genes and 10,156 noncoding RNA genes. We confirmed three known and identified one novel genome rearrangements. Our approach enables the rapid and scalable assembly of wheat genomes, the identification of structural variants, and the definition of complete gene models, all powerful resources for trait analysis and breeding of this key global crop.

Sex-specific changes in the aphid DNA methylation landscape.

Aphids present an ideal system to study epigenetics as they can produce diverse, but genetically identical, morphs in response to environmental stimuli. Here, using whole genome bisulphite sequencing and transcriptome sequencing of the green peach aphid (Myzus persicae), we present the first detailed analysis of cytosine methylation in an aphid and investigate differences in the methylation and transcriptional landscapes of male and asexual female morphs. We found that methylation primarily occurs in a CG dinucleotide (CpG) context and that exons are highly enriched for methylated CpGs, particularly at the 3' end of genes. Methylation is positively associated with gene expression, and methylated genes are more stably expressed than unmethylated genes. Male and asexual female morphs have distinct methylation profiles. Strikingly, these profiles are divergent between the sex chromosome and the autosomes; autosomal genes are hypomethylated in males compared to asexual females, whereas genes belonging to the sex chromosome, which is haploid in males, are hypermethylated. Overall, we found correlated changes in methylation and gene expression between males and asexual females, and this correlation was particularly strong for genes located on the sex chromosome. Our results suggest that differential methylation of sex-biased genes plays a role in aphid sexual differentiation.

Oakleaf: an S locus-linked mutation of Primula vulgaris that affects leaf and flower development.

In Primula vulgaris outcrossing is promoted through reciprocal herkogamy with insect-mediated cross-pollination between pin and thrum form flowers. Development of heteromorphic flowers is coordinated by genes at the S locus. To underpin construction of a genetic map facilitating isolation of these S locus genes, we have characterised Oakleaf, a novel S locus-linked mutant phenotype. We combine phenotypic observation of flower and leaf development, with classical genetic analysis and next-generation sequencing to address the molecular basis of Oakleaf. Oakleaf is a dominant mutation that affects both leaf and flower development; plants produce distinctive lobed leaves, with occasional ectopic meristems on the veins. This phenotype is reminiscent of overexpression of Class I KNOX-homeodomain transcription factors. We describe the structure and expression of all eight P. vulgaris PvKNOX genes in both wild-type and Oakleaf plants, and present comparative transcriptome analysis of leaves and flowers from Oakleaf and wild-type plants. Oakleaf provides a new phenotypic marker for genetic analysis of the Primula S locus. We show that none of the Class I PvKNOX genes are strongly upregulated in Oakleaf leaves and flowers, and identify cohorts of 507 upregulated and 314 downregulated genes in the Oakleaf mutant.

High-resolution transcriptional analysis of the regulatory influence of cell-to-cell signalling reveals novel genes that contribute to Xanthomonas phytopathogenesis.

The bacterium Xanthomonas campestris is an economically important pathogen of many crop species and a model for the study of bacterial phytopathogenesis. In X. campestris, a regulatory system mediated by the signal molecule DSF controls virulence to plants. The synthesis and recognition of the DSF signal depends upon different Rpf proteins. DSF signal generation requires RpfF whereas signal perception and transduction depends upon a system comprising the sensor RpfC and regulator RpfG. Here we have addressed the action and role of Rpf/DSF signalling in phytopathogenesis by high-resolution transcriptional analysis coupled to functional genomics. We detected transcripts for many genes that were unidentified by previous computational analysis of the genome sequence. Novel transcribed regions included intergenic transcripts predicted as coding or non-coding as well as those that were antisense to coding sequences. In total, mutation of rpfF, rpfG and rpfC led to alteration in transcript levels (more than fourfold) of approximately 480 genes. The regulatory influence of RpfF and RpfC demonstrated considerable overlap. Contrary to expectation, the regulatory influence of RpfC and RpfG had limited overlap, indicating complexities of the Rpf signalling system. Importantly, functional analysis revealed over 160 new virulence factors within the group of Rpf-regulated genes.

Genomics and biochemical analyses reveal a metabolon key to ß-L-ODAP biosynthesis in Lathyrus sativus.

Grass pea (Lathyrus sativus L.) is a rich source of protein cultivated as an insurance crop in Ethiopia, Eritrea, India, Bangladesh, and Nepal. Its resilience to both drought and flooding makes it a promising crop for ensuring food security in a changing climate. The lack of genetic resources and the crop's association with the disease neurolathyrism have limited the cultivation of grass pea. Here, we present an annotated, long read-based assembly of the 6.5 Gbp L. sativus genome. Using this genome sequence, we have elucidated the biosynthetic pathway leading to the formation of the neurotoxin, ß-L-oxalyl-2,3-diaminopropionic acid (ß-L-ODAP). The final reaction of the pathway depends on an interaction between L. sativus acyl-activating enzyme 3 (LsAAE3) and a BAHD-acyltransferase (LsBOS) that form a metabolon activated by CoA to produce ß-L-ODAP. This provides valuable insight into the best approaches for developing varieties which produce substantially less toxin.

Chromosome-scale genome assembly provides insights into rye biology, evolution and agronomic potential.

Rye (Secale cereale L.) is an exceptionally climate-resilient cereal crop, used extensively to produce improved wheat varieties via introgressive hybridization and possessing the entire repertoire of genes necessary to enable hybrid breeding. Rye is allogamous and only recently domesticated, thus giving cultivated ryes access to a diverse and exploitable wild gene pool. To further enhance the agronomic potential of rye, we produced a chromosome-scale annotated assembly of the 7.9-gigabase rye genome and extensively validated its quality by using a suite of molecular genetic resources. We demonstrate applications of this resource with a broad range of investigations. We present findings on cultivated rye's incomplete genetic isolation from wild relatives, mechanisms of genome structural evolution, pathogen resistance, low-temperature tolerance, fertility control systems for hybrid breeding and the yield benefits of rye-wheat introgressions.

Efficient and accurate detection of splice junctions from RNA-seq with Portcullis.

Next-generation sequencing technologies enable rapid and cheap genome-wide transcriptome analysis, providing vital information about gene structure, transcript expression, and alternative splicing. Key to this is the accurate identification of exon-exon junctions from RNA sequenced (RNA-seq) reads. A number of RNA-seq aligners capable of splitting reads across these splice junctions (SJs) have been developed; however, it has been shown that while they correctly identify most genuine SJs available in a given sample, they also often produce large numbers of incorrect SJs. Here, we describe the extent of this problem using popular RNA-seq mapping tools and present a new method, called Portcullis, to rapidly filter false SJs derived from spliced alignments. We show that Portcullis distinguishes between genuine and false-positive junctions to a high degree of accuracy across different species, samples, expression levels, error profiles, and read lengths. Portcullis is portable, efficient, and, to our knowledge, currently the only SJ prediction tool that reliably scales for use with large RNA-seq datasets and large, highly fragmented genomes, while delivering accurate SJs.

Leveraging multiple transcriptome assembly methods for improved gene structure annotation.

Background: The performance of RNA sequencing (RNA-seq) aligners and assemblers varies greatly across different organisms and experiments, and often the optimal approach is not known beforehand. Results: Here, we show that the accuracy of transcript reconstruction can be boosted by combining multiple methods, and we present a novel algorithm to integrate multiple RNA-seq assemblies into a coherent transcript annotation. Our algorithm can remove redundancies and select the best transcript models according to user-specified metrics, while solving common artifacts such as erroneous transcript chimerisms. Conclusions: We have implemented this method in an open-source Python3 and Cython program, Mikado, available on GitHub.

The ash dieback invasion of Europe was founded by two genetically divergent individuals.

Accelerating international trade and climate change make pathogen spread an increasing concern. Hymenoscyphus fraxineus, the causal agent of ash dieback, is a fungal pathogen that has been moving across continents and hosts from Asian to European ash. Most European common ash trees (Fraxinus excelsior) are highly susceptible to H. fraxineus, although a minority (~5%) have partial resistance to dieback. Here, we assemble and annotate a H. fraxineus draft genome, which approaches chromosome scale. Pathogen genetic diversity across Europe and in Japan, reveals a strong bottleneck in Europe, though a signal of adaptive diversity remains in key host interaction genes. We find that the European population was founded by two divergent haploid individuals. Divergence between these haplotypes represents the ancestral polymorphism within a large source population. Subsequent introduction from this source would greatly increase adaptive potential of the pathogen. Thus, further introgression of H. fraxineus into Europe represents a potential threat and Europe-wide biological security measures are needed to manage this disease.

Rapid transcriptional plasticity of duplicated gene clusters enables a clonally reproducing aphid to colonise diverse plant species.

BACKGROUND: The prevailing paradigm of host-parasite evolution is that arms races lead to increasing specialisation via genetic adaptation. Insect herbivores are no exception and the majority have evolved to colonise a small number of closely related host species. Remarkably, the green peach aphid, Myzus persicae, colonises plant species across 40 families and single M. persicae clonal lineages can colonise distantly related plants. This remarkable ability makes M. persicae a highly destructive pest of many important crop species. RESULTS: To investigate the exceptional phenotypic plasticity of M. persicae, we sequenced the M. persicae genome and assessed how one clonal lineage responds to host plant species of different families. We show that genetically identical individuals are able to colonise distantly related host species through the differential regulation of genes belonging to aphid-expanded gene families. Multigene clusters collectively upregulate in single aphids within two days upon host switch. Furthermore, we demonstrate the functional significance of this rapid transcriptional change using RNA interference (RNAi)-mediated knock-down of genes belonging to the cathepsin B gene family. Knock-down of cathepsin B genes reduced aphid fitness, but only on the host that induced upregulation of these genes. CONCLUSIONS: Previous research has focused on the role of genetic adaptation of parasites to their hosts. Here we show that the generalist aphid pest M. persicae is able to colonise diverse host plant species in the absence of genetic specialisation. This is achieved through rapid transcriptional plasticity of genes that have duplicated during aphid evolution.

wigExplorer, a BioJS component to visualise wig data.

wigExplorer is a BioJS component whose main purpose is to provide a platform for visualisation of wig-formatted data. Wig files are extensively used by genome browsers such as the UCSC Genome Browser. wigExplorer follows the BioJS standard specification, requiring a simple configuration and installation. wigExplorer provides an easy way to navigate the visible region of the canvas and allows interaction with other components via predefined events. Availability: http://github.com/biojs/biojs; http://dx.doi.org/10.5281/zenodo.7721.