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Abstract Background The garden pea, Pisum sativum, is among the best-investigated legume plants and of significant agro-commercial relevance. Pisum sativum has a large and complex genome and accordingly few comprehensive genomic resources exist. Results We analyzed the pea transcriptome at the highest possible amount of accuracy by current technology. We used next generation sequencing with the Roche/454 platform and evaluated and compared a variety of approaches, including diverse tissue libraries, normalization, alternative sequencing technologies, saturation estimation and diverse assembly strategies. We generated libraries from flowers, leaves, cotyledons, epi- and hypocotyl, and etiolated and light treated etiolated seedlings, comprising a total of 450 megabases. Libraries were assembled into 324,428 unigenes in a first pass assembly. A second pass assembly reduced the amount to 81,449 unigenes but caused a significant number of chimeras. Analyses of the assemblies identified the assembly step as a major possibility for improvement. By recording frequencies of Arabidopsis orthologs hit by randomly drawn reads and fitting parameters of the saturation curve we concluded that sequencing was exhaustive. For leaf libraries we found normalization allows partial recovery of expression strength aside the desired effect of increased coverage. Based on theoretical and biological considerations we concluded that the sequence reads in the database tagged the vast majority of transcripts in the aerial tissues. A pathway representation analysis showed the merits of sampling multiple aerial tissues to increase the number of tagged genes. All results have been made available as a fully annotated database in fasta format. Conclusions We conclude that the approach taken resulted in a high quality - dataset which serves well as a first comprehensive reference set for the model legume pea. We suggest future deep sequencing transcriptome projects of species lacking a genomics backbone will need to concentrate mainly on resolving the issues of redundancy and paralogy during transcriptome assembly.

Abstract Background Seagrasses are a polyphyletic group of monocotyledonous angiosperms that have adapted to a completely submerged lifestyle in marine waters. Here, we exploit two collections of expressed sequence tags (ESTs) of two wide-spread and ecologically important seagrass species, the Mediterranean seagrass Posidonia oceanica (L.) Delile and the eelgrass Zostera marina L., which have independently evolved from aquatic ancestors. This replicated, yet independent evolutionary history facilitates the identification of traits that may have evolved in parallel and are possible instrumental candidates for adaptation to a marine habitat. Results In our study, we provide the first quantitative perspective on molecular adaptations in two seagrass species. By constructing orthologous gene clusters shared between two seagrasses (Z. marina and P. oceanica) and eight distantly related terrestrial angiosperm species, 51 genes could be identified with detection of positive selection along the seagrass branches of the phylogenetic tree. Characterization of these positively selected genes using KEGG pathways and the Gene Ontology uncovered that these genes are mostly involved in translation, metabolism, and photosynthesis. Conclusions These results provide first insights into which seagrass genes have diverged from their terrestrial counterparts via an initial aquatic stage characteristic of the order and to the derived fully-marine stage characteristic of seagrasses. We discuss how adaptive changes in these processes may have contributed to the evolution towards an aquatic and marine existence.

Abstract Background The phloem of dicotyledonous plants contains specialized P-proteins (phloem proteins) that accumulate during sieve element differentiation and remain parietally associated with the cisternae of the endoplasmic reticulum in mature sieve elements. Wounding causes P-protein filaments to accumulate at the sieve plates and block the translocation of photosynthate. Specialized, spindle-shaped P-proteins known as forisomes that undergo reversible calcium-dependent conformational changes have evolved exclusively in the Fabaceae. Recently, the molecular characterization of three genes encoding forisome components in the model legume Medicago truncatula (MtSEO1, MtSEO2 and MtSEO3; SEO = sieve element occlusion) was reported, but little is known about the molecular characteristics of P-proteins in non-Fabaceae. Results We performed a comprehensive genome-wide comparative analysis by screening the M. truncatula, Glycine max, Arabidopsis thaliana, Vitis vinifera and Solanum phureja genomes, and a Malus domestica EST library for homologs of MtSEO1, MtSEO2 and MtSEO3 and identified numerous novel SEO genes in Fabaceae and even non-Fabaceae plants, which do not possess forisomes. Even in Fabaceae some SEO genes appear to not encode forisome components. All SEO genes have a similar exon-intron structure and are expressed predominantly in the phloem. Phylogenetic analysis revealed the presence of several subgroups with Fabaceae-specific subgroups containing all of the known as well as newly identified forisome component proteins. We constructed Hidden Markov Models that identified three conserved protein domains, which characterize SEO proteins when present in combination. In addition, one common and three subgroup specific protein motifs were found in the amino acid sequences of SEO proteins. SEO genes are organized in genomic clusters and the conserved synteny allowed us to identify several M. truncatula vs G. max orthologs as well as paralogs within the G. max genome. Conclusions The unexpected occurrence of forisome-like genes in non-Fabaceae plants may indicate that these proteins encode species-specific P-proteins, which is backed up by the phloem-specific expression profiles. The conservation of gene structure, the presence of specific motifs and domains and the genomic synteny argue for a common phylogenetic origin of forisomes and other P-proteins.

Abstract Background Creating new protein domain arrangements is a frequent mechanism of evolutionary innovation. While some domains always form the same combinations, others form many different arrangements. This ability, which is often referred to as versatility or promiscuity of domains, its a random evolutionary model in which a domain's promiscuity is based on its relative frequency of domains. Results We show that there is a clear relationship across genomes between the promiscuity of a given domain and its frequency. However, the strength of this relationship differs for different domains. We thus redefine domain promiscuity by defining a new index, DV I ("domain versatility index"), which eliminates the effect of domain frequency. We explore links between a domain's versatility, when unlinked from abundance, and its biological properties. Conclusion Our results indicate that domains occurring as single domain proteins and domains appearing frequently at protein termini have a higher DV I. This is consistent with previous observations that the evolution of domain re-arrangements is primarily driven by fusion of pre-existing arrangements and single domains as well as loss of domains at protein termini. Furthermore, we studied the link between domain age, defined as the first appearance of a domain in the species tree, and the DV I. Contrary to previous studies based on domain promiscuity, it seems as if the DV I is age independent. Finally, we find that contrary to previously reported findings, versatility is lower in Eukaryotes. In summary, our measure of domain versatility indicates that a random attachment process is sufficient to explain the observed distribution of domain arrangements and that several views on domain promiscuity need to be revised.

Abstract Background The evolution of complex molecular traits such as disulphide bridges often requires multiple mutations. The intermediate steps in such evolutionary trajectories are likely to be selectively neutral or deleterious. Therefore, large populations and long times may be required to evolve such traits. Results We propose that errors in transcription and translation may allow selection for the intermediate mutations, if the final trait provides a large enough selective advantage. We test this hypothesis using a population based model of protein evolution. Conclusion If an individual acquires one of two mutations needed for a novel trait, the second mutation can be introduced into the phenotype due to transcription and translation errors. If the novel trait is advantageous enough, the allele with only one mutation will spread through the population, even though the gene sequence does not yet code for the complettrait. Thus, errors allow protein sequences to "look-ahead" for a more direct path to a complex trait. Reviewers This article was reviewed by Eugene Koonin, Subhajyoti De (nomimated by Madan Babu), and David Krakauer.

Abstract Background bZIPs are transcription factors that are found throughout the eukarya from fungi to flowering plants and mammals. They contain highly conserved basic region (BR) and leucine zipper (LZ) domains and often function as environmental sensors. Specifically, bZIPs frequently have a role in mediating the response to oxidative stress, a crucial environmental signal that needs to be transduced to the gene regulatory network. Results Based on sequence comparisons and experimental data on a number of important bZIP transcription factors, we predict which bZIPs are under redox control and which are regulated via protein phosphorylation. By integrating genomic, phylogenetic and functional data from the literature, we then propose a link between oxidative stress and the choice of interaction partners for the bZIP proteins. Conclusion This integration permits the bZIP dimerization network to be interpreted in functional terms, especially in the context of the role of bZIP proteins in the response to environmental stress. This analysis demonstrates the importance of abiotic factors in shaping regulatory networks.

Protein coding features can emerge de novo in non coding transcripts, resulting in emergence of new protein coding genes. Studies across many species show that a large fraction of evolutionarily novel non-coding RNAs have an antisense overlap with protein coding genes. The open reading frames (ORFs) in these antisense RNAs could also overlap with existing ORFs. In this study, we investigate how the evolution an ORF could be constrained by its overlap with an existing ORF in three different reading frames. Using a combination of mathematical modeling and genome/transcriptome data analysis in two different model organisms, we show that antisense overlap can increase the likelihood of ORF emergence and reduce the likelihood of ORF loss, especially in one of the three reading frames. In addition to rationalising the repeatedly reported prevalence of de novo emerged genes in antisense transcripts, our work also provides a generic modeling and an analytical framework that can be used to understand evolution of antisense genes., (© 2024. The Author(s).)

For protein coding genes to emerge de novo from a non-genic DNA, the DNA sequence must gain an open reading frame (ORF) and the ability to be transcribed. The newborn de novo gene can further evolve to accumulate changes in its sequence. Consequently, it can also elongate or shrink with time. Existing literature shows that older de novo genes have longer ORF, but it is not clear if they elongated with time or remained of the same length since their inception. To address this question we developed a mathematical model of ORF elongation as a Markov-jump process, and show that ORFs tend to keep their length in short evolutionary timescales. We also show that if change occurs it is likely to be a truncation. Our genomics and transcriptomics data analyses of seven Drosophila melanogaster populations are also in agreement with the model's prediction. We conclude that selection could facilitate ORF length extension that may explain why longer ORFs were observed in old de novo genes in studies analysing longer evolutionary time scales. Alternatively, shorter ORFs may be purged because they may be less likely to yield functional proteins., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

De novo genes emerge from noncoding regions of genomes via succession of mutations. Among others, such mutations activate transcription and create a new open reading frame (ORF). Although the mechanisms underlying ORF emergence are well documented, relatively little is known about the mechanisms enabling new transcription events. Yet, in many species a continuum between absent and very prominent transcription has been reported for essentially all regions of the genome. In this study, we searched for de novo transcripts by using newly assembled genomes and transcriptomes of seven inbred lines of Drosophila melanogaster, originating from six European and one African population. This setup allowed us to detect sample specific de novo transcripts, and compare them to their homologous nontranscribed regions in other samples, as well as genic and intergenic control sequences. We studied the association with transposable elements (TEs) and the enrichment of transcription factor motifs upstream of de novo emerged transcripts and compared them with regulatory elements. We found that de novo transcripts overlap with TEs more often than expected by chance. The emergence of new transcripts correlates with regions of high guanine-cytosine content and TE expression. Moreover, upstream regions of de novo transcripts are highly enriched with regulatory motifs. Such motifs are more enriched in new transcripts overlapping with TEs, particularly DNA TEs, and are more conserved upstream de novo transcripts than upstream their 'nontranscribed homologs'. Overall, our study demonstrates that TE insertion is important for transcript emergence, partly by introducing new regulatory motifs from DNA TE families., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Apes possess two sex chromosomes-the male-specific Y chromosome and the X chromosome, which is present in both males and females. The Y chromosome is crucial for male reproduction, with deletions being linked to infertility 1 . The X chromosome is vital for reproduction and cognition 2 . Variation in mating patterns and brain function among apes suggests corresponding differences in their sex chromosomes. However, owing to their repetitive nature and incomplete reference assemblies, ape sex chromosomes have been challenging to study. Here, using the methodology developed for the telomere-to-telomere (T2T) human genome, we produced gapless assemblies of the X and Y chromosomes for five great apes (bonobo (Pan paniscus), chimpanzee (Pan troglodytes), western lowland gorilla (Gorilla gorilla gorilla), Bornean orangutan (Pongo pygmaeus) and Sumatran orangutan (Pongo abelii)) and a lesser ape (the siamang gibbon (Symphalangus syndactylus)), and untangled the intricacies of their evolution. Compared with the X chromosomes, the ape Y chromosomes vary greatly in size and have low alignability and high levels of structural rearrangements-owing to the accumulation of lineage-specific ampliconic regions, palindromes, transposable elements and satellites. Many Y chromosome genes expand in multi-copy families and some evolve under purifying selection. Thus, the Y chromosome exhibits dynamic evolution, whereas the X chromosome is more stable. Mapping short-read sequencing data to these assemblies revealed diversity and selection patterns on sex chromosomes of more than 100 individual great apes. These reference assemblies are expected to inform human evolution and conservation genetics of non-human apes, all of which are endangered species., (© 2024. The Author(s).)

De novo genes emerge from previously noncoding stretches of the genome. Their encoded de novo proteins are generally expected to be similar to random sequences and, accordingly, with no stable tertiary fold and high predicted disorder. However, structural properties of de novo proteins and whether they differ during the stages of emergence and fixation have not been studied in depth and rely heavily on predictions. Here we generated a library of short human putative de novo proteins of varying lengths and ages and sorted the candidates according to their structural compactness and disorder propensity. Using Förster resonance energy transfer combined with Fluorescence-activated cell sorting, we were able to screen the library for most compact protein structures, as well as most elongated and flexible structures. We find that compact de novo proteins are on average slightly shorter and contain lower predicted disorder than less compact ones. The predicted structures for most and least compact de novo proteins correspond to expectations in that they contain more secondary structure content or higher disorder content, respectively. Our experiments indicate that older de novo proteins have higher compactness and structural propensity compared with young ones. We discuss possible evolutionary scenarios and their implications underlying the age-dependencies of compactness and structural content of putative de novo proteins., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Most of the transcribed eukaryotic genomes are composed of non-coding transcripts. Among these transcripts, some are newly transcribed when compared to outgroups and are referred to as de novo transcripts. De novo transcripts have been shown to play a major role in genomic innovations. However, little is known about the rates at which de novo transcripts are gained and lost in individuals of the same species. Here, we address this gap and estimate the de novo transcript turnover rate with an evolutionary model. We use DNA long reads and RNA short reads from seven geographically remote samples of inbred individuals of Drosophila melanogaster to detect de novo transcripts that are gained on a short evolutionary time scale. Overall, each sampled individual contains around 2500 unspliced de novo transcripts, with most of them being sample specific. We estimate that around 0.15 transcripts are gained per year, and that each gained transcript is lost at a rate around 5× 10-5 per year. This high turnover of transcripts suggests frequent exploration of new genomic sequences within species. These rate estimates are essential to comprehend the process and timescale of de novo gene birth., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Genome sequencing quality, in terms of both read length and accuracy, is constantly improving. By combining long-read sequencing technologies with various scaffolding techniques, chromosome-level genome assemblies are now achievable at an affordable price for non-model organisms. Insects represent an exciting taxon for studying the genomic underpinnings of evolutionary innovations, due to ancient origins, immense species-richness, and broad phenotypic diversity. Here we summarize some of the most important methods for carrying out a comparative genomics study on insects. We describe available tools and offer concrete tips on all stages of such an endeavor from DNA extraction through genome sequencing, annotation, and several evolutionary analyses. Along the way we describe important insect-specific aspects, such as DNA extraction difficulties or gene families that are particularly difficult to annotate, and offer solutions. We describe results from several examples of comparative genomics analyses on insects to illustrate the fascinating questions that can now be addressed in this new age of genomics research., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Genome sequencing enables answering fundamental questions about the genetic basis of adaptation, population structure and epigenetic mechanisms. Yet, we usually need a suitable reference genome for mapping population-level resequencing data. In some model systems, multiple reference genomes are available, giving the challenging task of determining which reference genome best suits the data. Here, we compared the use of two different reference genomes for the three-spined stickleback (Gasterosteus aculeatus), one novel genome derived from a European gynogenetic individual and the published reference genome of a North American individual. Specifically, we investigated the impact of using a local reference versus one generated from a distinct lineage on several common population genomics analyses. Through mapping genome resequencing data of 60 sticklebacks from across Europe and North America, we demonstrate that genetic distance among samples and the reference genomes impacts downstream analyses. Using a local reference genome increased mapping efficiency and genotyping accuracy, effectively retaining more and better data. Despite comparable distributions of the metrics generated across the genome using SNP data (i.e. π, Tajima's D and F ST ), window-based statistics using different references resulted in different outlier genes and enriched gene functions. A marker-based analysis of DNA methylation distributions had a comparably high overlap in outlier genes and functions, yet with distinct differences depending on the reference genome. Overall, our results highlight how using a local reference genome decreases reference bias to increase confidence in downstream analyses of the data. Such results have significant implications in all reference-genome-based population genomic analyses., (© 2023 The Authors. Molecular Ecology Resources published by John Wiley & Sons Ltd.)