The field of genetics of bird migration advances, driven by exponential refinements of sequencing and tracking technologies. In willow warblers (Phylloscopus trochilus), a complex repeat-rich region named MARB (Migration Associated Repeat Block) has recently been found to correlate with the routes taken by individual birds from Europe to their African wintering grounds. However, the genomic location of this region remains unknown. Here, we characterized MARB using a combination of approaches to understand how it evolved. We describe the region using long-read genome assemblies of two willow warbler subspecies (P. t. trochilus and P. t. acredula), two related species, the common chiffchaff (P. collybita) and the greenish warbler (P. trochiloides), and whole genome sequencing data from 76 willow warblers. Finally, we applied karyotyping and fluorescent in situ hybridization techniques on willow warbler spermatocytes to cytogenetically locate MARB. Due to the many repeats, we cannot order scaffolds in silico, but probe hybridization on the karyotype shows that MARB constitutes a single locus (~27.5 Mb) spanning most of the 11th largest chromosome in the willow warbler genome. Interestingly, the MARB regions of all species share several characteristics such as relatively high GC content (50%), a high density of specific repeat families and notably, more than 800 olfactory receptor sequences. Regions homologous to MARB may exist in several migrant bird genomes, though currently unassembled due to their complexity. Resolving these in species with similar migratory polymorphisms to willow warblers will be essential to determine whether MARB influences migratory behaviour across species.

Despite technological advances in both tracking and sequencing technologies, finding the genetic mechanisms behind migratory traits remains a challenge. Recent studies have shown that migratory direction in European willow warblers (Phylloscopus trochilus) is mainly influenced by a repeat-rich region named MARB. Whether this genetic basis for orientation is unique to the willow warbler or instead shared by other closely related species is currently unknown. Therefore, here we investigate a close relative, the common chiffchaff (Phylloscopus collybita) by combining whole-genome resequencing data and geolocator tracks from two subspecies, P. c. collybita and P. c. abietinus. As in willow warblers, our geolocator data reveals that the two chiffchaff subspecies differ in orientation, with westward and eastward migratory directions from their breeding grounds in Sweden and south of the Baltics. Genome-wide divergence between the subspecies is low and consistent with a recent split within the last 100,000 years, as provided by demographic analyses. The divergence between subspecies is likely a result of shifts in allele frequencies across many loci although signals of differentiation are concentrated in the Z chromosome. Most differences, however, are found in the MARB region, raising the possibility that MARB contributes to orientation in chiffchaffs and may have evolved in parallel to willow warblers. Given the repeat-rich structure of this region, its characterization using short-read NGS sequencing remains challenging, and the diversity and nature of MARB haplotypes must be interpreted with caution.

Abstract Background The Norwegian lemming (Lemmus lemmus) is a small rodent endemic to the Fennoscandian alpine and arctic tundra. The species is known for cyclic population outbreaks and mass movements during peak years. Previous research based on microsatellites revealed high genetic variation but a weak population structure in the Norwegian lemming. Results In this study, we revisit the population structure of the species using genome-wide data. To do this, we generated a high-quality de novo reference genome for Lemmus lemmus, and resequenced genomes to 2.5–5 × coverage, from 86 lemmings sampled across the species’ entire geographic distribution. Our results reveal that the population is geographically structured into distinct subpopulations, with an overall pattern characterised by isolation-by-distance among subpopulations. Furthermore, our results are consistent with earlier work suggesting that the species survived the last ice age within a northern refugium. Conclusions Together, these findings provide a genome-wide perspective on today’s population structure of the Norwegian lemming. In addition, we provide a de novo reference genome, which we believe will be a valuable resource to the research community.

Distyly, an example of convergent evolution, is governed by a supergene, the S-locus, in several species. Recent studies highlight similar genomic architectures of independently evolved S-loci, but its mode of origin and whether similar regulatory pathways underlie the convergent evolution of distyly remains unclear. We examined the evolution of supergenes and mechanisms underlying distyly in Linum species that diverged c. 33 million years ago (Ma). Using haplotype-resolved genomes and population genomics, we identified and characterized the S-loci of Linum perenne (distylous) and Linum grandiflorum (style length dimorphic), and compared them to that of Linum tenue (distylous). We then tested for a conserved hormonal mechanism regulating style length polymorphism in Linum. The S-locus supergene was consistently hemizygous in short-styled individuals across all three species, although it showed variation in size, gene content, repeat elements and extent of recombination suppression. Two S-linked candidate genes, TSS1 (style length) and WDR-44 (anther height/pollen self-incompatibility), were conserved. Consistent with a brassinosteroid-dependent role of TSS1, epibrassinolide treatment revealed a conserved, morph-specific effect on style length. S-locus structural polymorphism, candidate distyly genes and mechanisms regulating style length remain conserved > 30 Ma in Linum. In combination with findings from other systems, our results suggest that the brassinosteroid pathway frequently contributes to style length polymorphism.

The European green toad (Bufotes viridis) is geographically widely distributed. While the species global conservation status is labeled as of least concern by the IUCN, it is declining in many parts of its range where populations are fragmented and isolated. A high-quality reference genome is an important resource for conservation genomic researchers who are trying to understand and interpret the genomic signals of population decline, inbreeding, and the accumulation of deleterious mutations. Here, we assembled and annotated a chromosome-level reference genome for B. viridis as part of the European Reference Genome Atlas pilot project. The genome assembly, with a size of ∼3.89 Gb consists of 11 chromosomes and an additional 2,096 unplaced scaffolds. The final assembly had a scaffold N50 value of 478.39 Mb and covered 90.4% single copy tetrapod orthologs, and 46.7% repetitive elements. Finally, a total of 23,830 protein-coding genes matching a known gene, together with 56,974 mRNAs were predicted. This high-quality reference genome will benefit amphibian evolutionary genomics research and enable conservation genetic studies to inform practical conservation work on this species.

Abstract Sponges are among the earliest branching extant animals. As such, genetic data from this group are valuable for understanding the evolution of various traits and processes in other animals. However, like many marine organisms, they are notoriously difficult to sequence, and hence, genomic data are scarce. Here, we present the draft genome assembly for the North Atlantic deep-sea high microbial abundance species Geodia barretti Bowerbank 1858, from a single individual collected on the West Coast of Sweden. The nuclear genome assembly has 4,535 scaffolds, an N50 of 48,447 bp and a total length of 144 Mb; the mitochondrial genome is 17,996 bp long. BUSCO completeness was 71.5%. The genome was annotated using a combination of ab initio and evidence-based methods finding 31,884 protein-coding genes.

ABSTRACT Chromatin-associated RNAs (caRNAs) modulate chromatin organization and function. The RNA exosome degrades different types of nuclear transcripts, but its role in chromatin has not been addressed. Here we have used Drosophila melanogaster S2 cells as a model system to identify the repertoire of caRNAs and establish the role of the exosome in their regulation. We have analyzed both unique and repetitive sequences, and combining RNA-seq and ATAC-seq we show that the simultaneous depletion of the exosome catalytic subunits RRP6 and DIS3 not only affects caRNA levels but also changes the local chromatin accessibility at specific loci. We have identified a group of exosome-sensitive genes that are involved in developmental regulation and are characterized by a balanced chromatin state in which Polycomb and Trithorax factors coexist. Our results reveal that RNA degradation by the exosome is an important mechanism for the homeostasis of such balanced chromatin states. Given that eukaryotic genomes are repetitive to a large extent, we have also analyzed repetitive caRNAs (rep-caRNAs) and we show that the exosome is needed to control repcaRNA levels and to maintain the degree of chromatin packaging in repetitive genomic regions. This role is particularly relevant in the pericentromeric regions where the exosome is required to silence LTR elements and maintain centromere organization.

SUMMARY Wind, rain, herbivores, obstacles, neighbouring plants, etc. provide important mechanical cues to steer plant growth and survival. Mechanostimulation to stimulate yield and stress resistance of crops is of significant research interest, yet a molecular understanding of transcriptional responses to touch is largely absent in cereals. To address this, we performed whole‐genome transcriptomics following mechanostimulation of wheat, barley, and the recent genome‐sequenced oat. The largest transcriptome changes occurred ±25 min after touching, with most of the genes being upregulated. While most genes returned to basal expression level by 1–2 h in oat, many genes retained high expression even 4 h post‐treatment in barley and wheat. Functional categories such as transcription factors, kinases, phytohormones, and Ca 2+ regulation were affected. In addition, cell wall‐related genes involved in (hemi)cellulose, lignin, suberin, and callose biosynthesis were touch‐responsive, providing molecular insight into mechanically induced changes in cell wall composition. Furthermore, several cereal‐specific transcriptomic footprints were identified that were not observed in Arabidopsis. In oat and barley, we found evidence for systemic spreading of touch‐induced signalling. Finally, we provide evidence that both the jasmonic acid‐dependent and the jasmonic acid‐independent pathways underlie touch‐signalling in cereals, providing a detailed framework and marker genes for further study of (a)biotic stress responses in cereals.

The major histocompatibility complex (MHC) is of central importance to the immune system, and an optimal MHC diversity is believed to maximize pathogen elimination. Birds show substantial variation in MHC diversity, ranging from few genes in most bird orders to very many genes in passerines. Our understanding of the evolutionary trajectories of the MHC in passerines is hampered by lack of data on genomic organization. Therefore, we assembled and annotated the MHC genomic region of the great reed warbler (Acrocephalus arundinaceus), using long-read sequencing and optical mapping. The MHC region is large (>5.5 Mb), characterized by structural changes compared to hitherto investigated bird orders and shows higher repeat content than the genome average. These features were supported by analyses in three additional passerines. MHC genes in passerines are found in two different chromosomal arrangements, either as single copy MHC genes located among non-MHC genes, or as tandemly duplicated tightly linked MHC genes. Some single copy MHC genes are old and putative orthologues among species. In contrast tandemly duplicated MHC genes are monophyletic within species and have evolved by simultaneous gene duplication of several MHC genes. Structural differences in the MHC genomic region among bird orders seem substantial compared to mammals and have possibly been fuelled by clade-specific immune system adaptations. Our study provides methodological guidance in characterizing complex genomic regions, constitutes a resource for MHC research in birds, and calls for a revision of the general belief that avian MHC has a conserved gene order and small size compared to mammals.

How the avian sex chromosomes first evolved from autosomes remains elusive as 100 million years (My) of divergence and degeneration obscure their evolutionary history. The Sylvioidea group of songbirds is interesting for understanding avian sex chromosome evolution because a chromosome fusion event ∼24 Ma formed "neo-sex chromosomes" consisting of an added (new) and an ancestral (old) part. Here, we report the complete female genome (ZW) of one Sylvioidea species, the great reed warbler (Acrocephalus arundinaceus). Our long-read assembly shows that the added region has been translocated to both Z and W, and whereas the added-Z has retained its gene order the added-W part has been heavily rearranged. Phylogenetic analyses show that recombination between the homologous added-Z and -W regions continued after the fusion event, and that recombination suppression across this region took several million years to be completed. Moreover, recombination suppression was initiated across multiple positions over the added-Z, which is not consistent with a simple linear progression starting from the fusion point. As expected following recombination suppression, the added-W show signs of degeneration including repeat accumulation and gene loss. Finally, we present evidence for nonrandom maintenance of slowly evolving and dosage-sensitive genes on both ancestral- and added-W, a process causing correlated evolution among orthologous genes across broad taxonomic groups, regardless of sex linkage.

Background Sex chromosomes have evolved independently multiple times in eukaryotes and are therefore considered a prime example of convergent genome evolution. Sex chromosomes are known to emerge after recombination is halted between a homologous pair of chromosomes, and this leads to a range of non-adaptive modifications causing gradual degeneration and gene loss on the sex-limited chromosome. However, the proximal causes of recombination suppression and the pace at which degeneration subsequently occurs remain unclear. Results Here, we use long- and short-read single-molecule sequencing approaches to assemble and annotate a draft genome of the basket willow,Salix viminalis, a species with a female heterogametic system at the earliest stages of sex chromosome emergence. Our single-molecule approach allowed us to phase the emerging Z and W haplotypes in a female, and we detected very low levels of Z/W single-nucleotide divergence in the non-recombining region. Linked-read sequencing of the same female and an additional male (ZZ) revealed the presence of two evolutionary strata supported by both divergence between the Z and W haplotypes and by haplotype phylogenetic trees. Gene order is still largely conserved between the Z and W homologs, although the W-linked region contains genes involved in cytokinin signaling regulation that are not syntenic with the Z homolog. Furthermore, we find no support across multiple lines of evidence for inversions, which have long been assumed to halt recombination between the sex chromosomes. Conclusions Our data suggest that selection against recombination is a more gradual process at the earliest stages of sex chromosome formation than would be expected from an inversion and may result instead from the accumulation of transposable elements. Our results present a cohesive understanding of the earliest genomic consequences of recombination suppression as well as valuable insights into the initial stages of sex chromosome formation and regulation of sex differentiation.

ABSTRACT How the avian sex chromosomes first evolved from autosomes remains elusive as 100 million years (Myr) of divergence and degeneration obscure their evolutionary history. Sylvioidea songbirds is an emerging model for understanding avian sex chromosome evolution because a unique chromosome fusion event ∼24 Myr ago has formed enlarged “neo-sex chromosomes” consisting of an added (new) and an ancestral (old) part. Here, we report the female genome (ZW) of one Sylvioidea species, the great reed warbler ( Acrocephalus arundinaceus ). We confirm that the added region has been translocated to both Z and W, and show that the added-W part has been heavily reorganised within itself and with the ancestral-W. Next, we show that recombination between Z and W continued after the fusion event, and that recombination suppression took ∼10 Myr to be completed and arose locally and non-linearly along the sex chromosomes. This pattern is inconsistent with that of large inversions and instead suggests gradual and mosaic recombination suppression. We find that the added-W mirrors the ancestral-W in terms of repeat accumulation, loss of genetic variation, and gene degeneration. Lastly, we show that genes being maintained on W are slowly evolving and dosage sensitive, and that highly conserved genes across broad taxonomic groups, regardless of sex-linkage, evolve slower on both Z and W. This study reveals complex expansion of recombination suppression along avian sex chromosomes, and that the evolutionary trajectory of sex-linked genes is highly predictable and governed partly by sex-linkage per se , partly by their functional properties.

Blue mold, caused by the necrotrophic fungal pathogen Penicillium expansum, causes serious postharvest losses in apple, and threatens human health through production of the potent mycotoxin patulin. Recent studies indicate a quantitative control of resistance against this disease in apple cultivars. A whole genome apple microarray covering 60k transcripts was used to identify gene(s) that appear to be differentially regulated between resistant and susceptible cultivars in P. expansum-infected fruits. A number of potential candidates was encountered among defense- and oxidative stress-related genes, cell wall modification and lignification genes, and genes related to localization and transport. Induction of one cell wall-related gene and three genes involved in the ‘down-stream’ flavonoid biosynthesis pathway, demonstrates the fundamental role of the cell wall as an important barrier, and suggests that fruit flavonoids are involved in the resistance to blue mold. Moreover, exogenous application of the plant hormone methyl jasmonate (MeJA) reduced the symptoms resulting from inoculating apples with P. expansum. This is the first report linking MeJA and activation of cell wall and flavonoid pathway genes to resistance against blue mold in a study comparing different cultivars of domesticated apple. Our results provide an initial categorization of genes that are potentially involved in the resistance mechanism, and should be useful for developing tools for gene marker-assisted breeding of apple cultivars with an improved resistance to blue mold.

A better understanding of transcriptional and post-transcriptional regulation of gene expression in bacteria relies on studying their transcriptome. RNA sequencing methods are used not only to assess RNA abundance but also the exact boundaries of primary and processed transcripts. Here, we developed a method, called identification of specific cleavage position (ISCP), which enables the identification of direct endoribonuclease targets in vivo by comparing the 5΄ and 3΄ ends of processed transcripts between wild type and RNase deficient strains. To demonstrate the ISCP method, we used as a model the double-stranded specific RNase III in the human pathogen Streptococcus pyogenes. We mapped 92 specific cleavage positions (SCPs) among which, 48 were previously described and 44 are new, with the characteristic 2 nucleotides 3΄ overhang of RNase III. Most SCPs were located in untranslated regions of RNAs. We screened for RNase III targets using transcriptomic differential expression analysis (DEA) and compared those with the RNase III targets identified using the ISCP method. Our study shows that in S. pyogenes, under standard growth conditions, RNase III has a limited impact both on antisense transcripts and on global gene expression with the expression of most of the affected genes being downregulated in an RNase III deletion mutant.

Lycopodiophyta-consisting of three orders, Lycopodiales, Isoetales and Selaginellales, with different types of shoot apical meristems (SAMs)-form the earliest branch among the extant vascular plants. They represent a sister group to all other vascular plants, from which they differ in that their leaves are microphylls-that is, leaves with a single, unbranched vein, emerging from the protostele without a leaf gap-not megaphylls. All leaves represent determinate organs originating on the flanks of indeterminate SAMs. Thus, leaf formation requires the suppression of indeterminacy, that is, of KNOX transcription factors. In seed plants, this is mediated by different groups of transcription factors including ARP and YABBY.We generated a shoot tip transcriptome of Huperzia selago (Lycopodiales) to examine the genes involved in leaf formation. Our H. selago transcriptome does not contain any ARP homolog, although transcriptomes of Selaginella spp. do. Surprisingly, we discovered a YABBY homolog, although these transcription factors were assumed to have evolved only in seed plants.The existence of a YABBY homolog in H. selago suggests that YABBY evolved already in the common ancestor of the vascular plants, and subsequently was lost in some lineages like Selaginellales, whereas ARP may have been lost in Lycopodiales. The presence of YABBY in the common ancestor of vascular plants would also support the hypothesis that this common ancestor had a simplex SAM. Furthermore, a comparison of the expression patterns of ARP in shoot tips of Selaginella kraussiana (Harrison CJ, etal. 2005. Independent recruitment of a conserved developmental mechanism during leaf evolution. Nature 434(7032):509-514.) and YABBY in shoot tips of H. selago implies that the development of microphylls, unlike megaphylls, does not seem to depend on the combined activities of ARP and YABBY. Altogether, our data show that Lycopodiophyta are a diverse group; so, in order to understand the role of Lycopodiophyta in evolution, representatives of Lycopodiales, Selaginellales, as well as of Isoetales, have to be examined.

Comparative transcriptomics between species can provide valuable understanding of plant-pathogen interactions. Here, we focus on wild Solanum species and potato clones with varying degree of resistance against Phytophthora infestans, which causes the devastating late blight disease in potato. The transcriptomes of three wild Solanum species native to Southern Sweden, Solanum dulcamara, Solanum nigrum, and Solanum physalifolium were compared to three potato clones, Desiree (cv.), SW93-1015 and Sarpo Mira. Desiree and S. physalifolium are susceptible to P. infestans whereas the other four have different degrees of resistance. By building transcript families based on de novo assembled RNA-seq across species and clones and correlating these to resistance phenotypes, we created a novel workflow to identify families with expanded or depleted number of transcripts in relation to the P. infestans resistance level. Analysis was facilitated by inferring functional annotations based on the family structure and semantic clustering. More transcript families were expanded in the resistant clones and species and the enriched functions of these were associated to expected gene ontology (GO) terms for resistance mechanisms such as hypersensitive response, host programmed cell death and endopeptidase activity. However, a number of unexpected functions and transcripts were also identified, for example transmembrane transport and protein acylation expanded in the susceptible group and a cluster of Zinc knuckle family proteins expanded in the resistant group. Over 400 expressed putative resistance (R-)genes were identified and resistant clones Sarpo Mira and SW93-1015 had ca 25% more expressed putative R-genes than susceptible cultivar Desiree. However, no differences in numbers of susceptibility (S-)gene homologs were seen between species and clones. In addition, we identified P. infestans transcripts including effectors in the early stages of P. infestans-Solanum interactions.

Potato is the third largest food crop in the world, however, the high degree of heterozygosity, the tetrasomic inheritance and severe inbreeding depression are major difficulties for conventional potato breeding. The rapid development of modern breeding methods offers new possibilities to enhance breeding efficiency and precise improvement of desirable traits. New site-directed mutagenesis techniques that can directly edit the target genes without any integration of recombinant DNA are especially favorable. Here we present a successful pipeline for site-directed mutagenesis in tetraploid potato through transient TALEN expression in protoplasts. The transfection efficiency of protoplasts was 38-39% and the site-directed mutation frequency was 7-8% with a few base deletions as the predominant type of mutation. Among the protoplast-derived calli, 11-13% showed mutations and a similar frequency (10%) was observed in the regenerated shoots. Our results indicate that the site-directed mutagenesis technology could be used as a new breeding method in potato as well as for functional analysis of important genes to promote sustainable potato production. (C) 2015 Elsevier B.V. All rights reserved.