The Asteraceae is the largest angiosperm family with more than 25,000 species. Individual studies have shown that MADS-box and TCP transcription factors are regulators of the development and symmetry of flowers, contributing to their iconic flower-head (capitulum) and floret. However, a systematic study of MADS-box and TCP genes across the Asteraceae is lacking. We performed a comparative analysis of genome sequences of 33 angiosperm species including our de novo assembly of diploid sexual dandelion (Taraxacum officinale) and 11 other Asteraceae to investigate the lineage-specific evolution of MADS-box and TCP genes in the Asteraceae. We compared the phylogenomic results of MADS-box and TCP genes with their expression in T. officinale floral tissues at different developmental stages to demonstrate the regulation of genes with Asteraceae-specific attributes. Here, we show that MADS-box MIKCc and TCP-CYCLOIDEA (CYC) genes have expanded in the Asteraceae. The phylogenomic analysis identified AGAMOUS-like (AG-like: SEEDSTICK [STK]-like), SEPALATA-like (SEP3-like), and TCP-PROLIFERATING CELL FACTOR (PCF)-like copies with lineage-specific genomic contexts in the Asteraceae, Cichorioideae, or dandelion. Different expression patterns of some of these gene copies suggest functional divergence. We also confirm the presence and revisit the evolutionary history of previously named "Asteraceae-Specific MADS-box genes (AS-MADS)." Specifically, we identify non-Asteraceae homologs, indicating a more ancient origin of this gene clade. Syntenic relationships support that AS-MADS is paralogous to FLOWERING LOCUS C (FLC) as demonstrated by the shared ancient duplication of FLC and SEP3.

Cap-snatching is a mechanism applied by segmented, negative strand (-) RNA viruses (NSVs) to initiate genome transcription. So far, the cap donor source of cytoplasmic-replicating NSVs has remained elusive. Recently, studies pointed to processing body (P body, PB) as the potential source for providing capped RNAs but conclusive evidence is still lacking. To attempt identifying these sources, here the 5' non-viral leader sequences of Tomato spotted wilt virus (TSWV) N mRNAs were analysed by high-throughput sequencing (HTS) from plants subjected to normal and heat-stress conditions, and subsequently mapped on host donor transcripts. The majority of non-viral heterogenous, host-derived leader sequences ranged in size between ~10-20 nt and contained A or AG residues at the cleavage site and the presence of certain sequence motifs. Mapping the capped-leader sequences to the 5' UTR region of genes encoded by the Nicotiana tabacum genome, identified 348 donor genes and which were specifically enriched in cellular photosynthesis pathway. Nineteen of those were clearly expressed differentially at normal condition versus heat-stress conditions. Although the results did not point towards snatching of capped-RNA leader sequences from certain cytoplasmic RNA granules in particular, they indicated photosynthesis downregulation (and development of disease symptoms) partially result from cap-snatching.

Background Adaptive laboratory evolution (ALE) is a powerful method for strain optimization towards abiotic stress factors and for identifying adaptation mechanisms. In this study, the green microalga Picochlorum sp. BPE23 was cultured under supra-optimal temperature to force genetic adaptation. The robustness and adaptive capacity of Picochlorum strains turned them into an emerging model for evolutionary studies on abiotic stressors such as temperature, salinity, and light. Results Mutant strains showed an expanded maximal growth temperature of 44.6 °C, whereas the maximal growth temperature of the wild-type strain was 42 °C. Moreover, at the optimal growth temperature of 38 °C, the biomass yield on light was 22.3% higher, and the maximal growth rate was 70.5% higher than the wild type. Genome sequencing and transcriptome analysis were performed to elucidate the mechanisms behind the improved phenotype. A de novo assembled phased reference genome allowed the identification of 21 genic mutations involved in various processes. Moreover, approximately half of the genome contigs were found to be duplicated or even triplicated in all mutants, suggesting a causal role in adaptation. Conclusions The developed tools and mutant strains provide a strong framework from whereupon Picochlorum sp. BPE23 can be further developed. Moreover, the extensive strain characterization provides evidence of how microalgae evolve to supra-optimal temperature and to photobioreactor growth conditions. With this study, microalgal evolutionary mechanisms were identified by combining ALE with genome sequencing.

Climate change will strongly affect the developmental timing of insects, as their development rate depends largely on ambient temperature. However, we know little about the genetic mechanisms underlying the temperature sensitivity of embryonic development in insects. We investigated embryonic development rate in the winter moth (Operophtera brumata), a species with egg dormancy which has been under selection due to climate change. We used RNA sequencing to investigate which genes are involved in the regulation of winter moth embryonic development rate in response to temperature. Over the course of development, we sampled eggs before and after an experimental change in ambient temperature, including two early development weeks when the temperature sensitivity of eggs is low and two late development weeks when temperature sensitivity is high. We found temperature-responsive genes that responded in a similar way across development, as well as genes with a temperature response specific to a particular development week. Moreover, we identified genes whose temperature effect size changed around the switch in temperature sensitivity of development rate. Interesting candidate genes for regulating the temperature sensitivity of egg development rate included genes involved in histone modification, hormonal signalling, nervous system development and circadian clock genes. The diverse sets of temperature-responsive genes we found here indicate that there are many potential targets of selection to change the temperature sensitivity of embryonic development rate. Identifying for which of these genes there is genetic variation in wild insect populations will give insight into their adaptive potential in the face of climate change.

This study reveals the genomic architecture of a rapidly evolving mutation which segregates as a single-locus, X-linked trait -- flatwing -- in wild Hawaiian field crickets (Teleogryllus oceanicus). Flatwingsilences males by eliminating sound-producing structures on their forewings. Silence protects them from an acoustically-orienting parasitoid fly (Ormia ochracea), but interferes with their ability to attract and court females for mating. Silent crickets spread rapidly on several Hawaiian islands under pressure from the flies, representing one of the fastest rates of evoutionary change documented in the wild. Here we present an annotated genome sequence of T. oceanicus along with a linkage map and QTL analysis of the trait derived from RAD-sequencing of a backcrossed mapping population. RNA-seq was used to probe the functional pathways affected by the mutation during early development, and pleiotropic effects on another signaling trait, cuticular hydrocarbons, were assessed and genetically mapped.

RNA-seq data collected from Teleogryllus oceanicus of silent and singing morphs at embryonic stages

Evolutionary adaptation is generally thought to occur through incremental mutational steps, but large mutational leaps can occur during its early stages. These are challenging to study in nature due to the difficulty of observing new genetic variants as they arise and spread, but characterizing their genomic dynamics is important for understanding factors favoring rapid adaptation. Here, we report genomic consequences of recent, adaptive song loss in a Hawaiian population of field crickets (Teleogryllus oceanicus). A discrete genetic variant, flatwing, appeared and spread approximately 15 years ago. Flatwing erases sound-producing veins on male wings. These silent flatwing males are protected from a lethal, eavesdropping parasitoid fly. We sequenced, assembled and annotated the cricket genome, produced a linkage map, and identified a flatwing quantitative trait locus covering a large region of the X chromosome. Gene expression profiling showed that flatwing is associated with extensive genome-wide effects on embryonic gene expression. We found that flatwing male crickets express feminized chemical pheromones. This male feminizing effect, on a different sexual signaling modality, is genetically associated with the flatwing genotype. Our findings suggest that the early stages of evolutionary adaptation to extreme pressures can be accompanied by greater genomic and phenotypic disruption than previously appreciated, and highlight how abrupt adaptation might involve suites of traits that arise through pleiotropy or genomic hitchhiking.

Secondary trait loss is widespread and has profound consequences, from generating diversity to driving adaptation. Sexual trait loss is particularly common. Its genomic impact is challenging to reconstruct because most reversals occurred in the distant evolutionary past and must be inferred indirectly, and questions remain about the extent of disruption caused by pleiotropy, altered gene expression and loss of homeostasis. We tested the genomic signature of recent sexual signal loss in Hawaiian field crickets, Teleogryllus oceanicus . Song loss is controlled by a sex-linked Mendelian locus, flatwing , which feminises male wings by erasing sound-producing veins. This variant spread rapidly under pressure from an eavesdropping parasitoid fly. We sequenced, assembled and annotated the T. oceanicus genome, produced a high-density linkage map, and localised flatwing on the X chromosome. We characterised pleiotropic effects of flatwing , including changes in embryonic gene expression and alteration of another sexual signal, chemical pheromones. Song loss is associated with pleiotropy, hitchhiking and genome-wide regulatory disruption which feminises flatwing male pheromones. The footprint of recent adaptive trait loss illustrates R. A. Fisher's influential prediction that variants with large mutational effect sizes can invade genomes during the earliest stages of adaptation to extreme pressures, despite having severely disruptive genomic consequences.

Nanopore sequencing is a novel approach to nucleic acid analysis that generates long, error-prone reads. Since device components, base calling software and best practices for sample preparation are updated frequently and extensively, the nature of the produced data also changes frequently. As a result, peer-reviewed publications on de novo assembly pipeline benchmarking efforts are quickly rendered outdated by the next major improvement to the sequencing platforms. To provide the user community with a faster, more flexible alternative to peer-reviewed benchmark papers for de novo assembly tool performance we constructed poreTally, a comprehensive benchmarking tool. poreTally automatically assembles a given read set using several often-used assembly pipelines, analyzes the resulting assemblies for correctness and continuity, and finally generates a quality report. Results can immediately be shared with peers in a Github/Gitlab repository. Furthermore, we aim to give a more inclusive overview of assembly pipeline performance than any individual research group can, by offering users the possibility to submit their results to a collective benchmarking effort. poreTally is available on Github.

The value of circulating microRNAs (miRNAs) as noninvasive biomarkers of human disease has been extensively demonstrated. Significant potential also exists in other species, particularly in relation to control of veterinary diseases and selection/monitoring of production traits in livestock. Although robust protocols have been developed for miRNA profiling of human biofluids, significant optimization may be required before these can be applied to other species. In this chapter, we describe protocols for small-RNA sequencing and RT-qPCR analyses of plasma samples from livestock species. In addition, we provide brief data analysis protocols for small-RNA sequencing and RT-qPCR data. Finally, we highlight important considerations for these protocols such as low RNA yield, platform-specific biases, and optimal normalization approaches.

Linking intraspecific and interspecific divergence is an important challenge in speciation research. X chromosomes are expected to evolve faster than autosomes and disproportionately contribute to reproductive barriers, and comparing genetic variation on X and autosomal markers within and between species can elucidate evolutionary processes that shape genome variation. We performed RADseq on a 16 population transect of two closely related Australian cricket species, Teleogryllus commodus and T. oceanicus, covering allopatry and sympatry. This classic study system for sexual selection provides a rare exception to Haldane's rule, as hybrid females are sterile. We found no evidence of recent introgression, despite the fact that the species coexist in overlapping habitats in the wild and interbreed in the laboratory. Putative X‐linked loci showed greater differentiation between species compared with autosomal loci. However, population differentiation within species was unexpectedly lower on X‐linked markers than autosomal markers, and relative X‐to‐autosomal genetic diversity was inflated above neutral expectations. Populations of both species showed genomic signatures of recent population expansions, but these were not strong enough to account for the inflated X/A diversity. Instead, most of the excess polymorphism on the X could better be explained by sex‐biased processes that increase the relative effective population size of the X, such as interspecific variation in the strength of sexual selection among males. Taken together, the opposing patterns of diversity and differentiation at X versus autosomal loci implicate a greater role for sex‐linked genes in maintaining species boundaries in this system.

Nanopore technology provides a novel approach to DNA sequencing that yields long, label-free reads of constant quality. The first commercial implementation of this approach, the MinION, has shown promise in various sequencing applications. This review gives an up-to-date overview of the MinION's utility as a de novo sequencing device. It is argued that the MinION may allow for portable and affordable de novo sequencing of even complex genomes in the near future, despite the currently error-prone nature of its reads. Through continuous updates to the MinION hardware and the development of new assembly pipelines, both sequencing accuracy and assembly quality have already risen rapidly. However, this fast pace of development has also lead to a lack of overview of the expanding landscape of analysis tools, as performance evaluations are outdated quickly. As the MinION is approaching a state of maturity, its user community would benefit from a thorough comparative benchmarking effort of de novo assembly pipelines in the near future. An earlier version of this article can be found on bioRxiv.

Nanopore technology provides a novel approach to DNA sequencing that yields long, label-free reads of constant quality. The first commercial implementation of this approach, the MinION, has shown promise in various sequencing applications. This review gives an up-to-date overview of the MinION's utility as a de novo sequencing device. It is argued that the MinION may allow for portable and affordable de novo sequencing of even complex genomes in the near future, despite the currently error-prone nature of its reads. Through continuous updates to the MinION hardware and the development of new assembly pipelines, both sequencing accuracy and assembly quality have already risen rapidly. However, this fast pace of development has also lead to a lack of overview of the expanding landscape of analysis tools, as performance evaluations are outdated quickly. As the MinION is approaching a state of maturity, its user community would benefit from a thorough comparative benchmarking effort of de novo assembly pipelines in the near future. An earlier version of this article can be found on bioRxiv.

Nanopore technology provides a novel approach to DNA sequencing that yields long, label-free reads of constant quality. The first commercial implementation of this approach, the MinION, has shown promise in various sequencing applications. The presented literature review gives an up-to-date overview of the MinION’s utility as a de novo sequencing device. It is argued that the MinION may allow for portable and affordable de novo sequencing of even complex genomes in the near future, despite the currently error-prone nature of its reads. Through continuous updates to the MinION hardware and the development of new assembly pipelines, both sequencing accuracy and assembly quality have already risen rapidly. However, this fast pace of development has also lead to a lack of oversight in the expanding landscape of analysis tools, as performance evaluations are outdated quickly. Now that the MinION is approaching a state of maturity, a thorough comparative benchmarking effort of de novo assembly pipelines may be at place.

Geographical variation among contiguous populations is frequently attributed to ecological divergence or historical isolation followed by secondary contact. Distinguishing between these effects is key to studies of incipient speciation and could be revealed by different genomic signatures. We used RAD-seq analyses to examine morphologically divergent populations of the endemic lizard (Gallotia galloti) from the volcanic island of Tenerife. Previous analyses have suggested ecological and historical causes to explain the morphological diversity. Analyses of 276,483 single nucleotide polymorphisms (SNPs) from >20 Mbp of the genome revealed one genetically divergent population from Anaga, a region associated with divergent mtDNA lineages in other Tenerife endemics. This population also has a high number of private alleles, and its divergence can be explained by historical isolation. Bayesian outlier analyses identified a small proportion of SNPs as candidates for selection (0.04%) which were strongly differentiated between xeric and mesic habitat types. Individual testing for specific xeric-mesic selection using an alternative approach also supported ecological divergence in a similarly small proportion of SNPs. The study indicates the roles of both historical isolation and ecological divergence in shaping genomic diversity in G. galloti. However, north-south morphological divergence appears solely associated with the latter and likely involves a relatively small proportion of the genome.

Motivation: The Oxford Nanopore MinION device represents a unique sequencing technology. As a mobile sequencing device powered by the USB port of a laptop, the MinION has huge potential applications. To enable these applications, the bioinformatics community will need to design and build a suite of tools specifically for MinION data. Results: Here we present poRe, a package for the statistical software R that enables users to manipulate, organize, summarise and visualize MinION nanopore sequencing data. As a packge for R, poRe has been tested on both Windows and Linux. Crucially, the Windows version allows users to analyse MinION data on the Windows laptop attached to the device Availability: Pre-built R packages for Windows and Linux are available under a BSD license at http://sourceforge.net/projects/rpore/ Contact: mick.watson@roslin.ed.ac.uk

Three strikingly different alternative male mating morphs (aggressive 'independents', semicooperative 'satellites' and female-mimic 'faeders') coexist as a balanced polymorphism in the ruff, Philomachus pugnax, a lek-breeding wading bird. Major differences in body size, ornamentation, and aggressive and mating behaviors are inherited as an autosomal polymorphism. We show that development into satellites and faeders is determined by a supergene consisting of divergent alternative, dominant and non-recombining haplotypes of an inversion on chromosome 11, which contains 125 predicted genes. Independents are homozygous for the ancestral sequence. One breakpoint of the inversion disrupts the essential CENP-N gene (encoding centromere protein N), and pedigree analysis confirms the lethality of homozygosity for the inversion. We describe new differences in behavior, testis size and steroid metabolism among morphs and identify polymorphic genes within the inversion that are likely to contribute to the differences among morphs in reproductive traits.

Bacteroides fragilis is a commensal member of human gut microbiota and an opportunistic pathogen. It is a major cause of life-threatening, anaerobic infections in soft tissue and abscesses. A characteristic of B. fragilis is within-strain variation in the genome. A range of invertible promoters and shufflons control expression of surface components and antigenically active large polysaccharides. This within-strain variation, coupled with repeat content, greatly complicates the assembly of the genome sequence from short reads. Short read data also make it difficult to assess the state of individual switches in the strain without direct expression data. We used a combination of Illumina shotgun reads and Oxford Nanopore MinION reads to generate a single scaffold genome assembly of a pathogenic B. fragilis strain BE1. We show that orientation of shufflons and invertible promotors can be assessed using only the long Nanopore MinION reads. This allows the characterisation of B. fragilis colonies in terms of expressed polysaccharides and facilitates in studying the changeable nature of this pathogen.

Background Second and third generation sequencing technologies have revolutionised bacterial genomics. Short-read Illumina reads result in cheap but fragmented assemblies, whereas longer reads are more expensive but result in more complete genomes. The Oxford Nanopore MinION device is a revolutionary mobile sequencer that can produce thousands of long, single molecule reads. Results We sequenced Bacteroides fragilis strain BE1 using both the Illumina MiSeq and Oxford Nanopore MinION platforms. We were able to assemble a single chromosome of 5.18 Mb, with no gaps, using publicly available software and commodity computing hardware. We identified gene rearrangements and the state of invertible promoters in the strain. Conclusions The single chromosome assembly of Bacteroides fragilis strain BE1 was achieved using only modest amounts of data, publicly available software and commodity computing hardware. This combination of technologies offers the possibility of ultra-cheap, high quality, finished bacterial genomes.