oskar acts with the transcription factor Creb to regulate long-term memory in crickets.

Novel genes have the potential to drive the evolution of new biological mechanisms, or to integrate into preexisting regulatory circuits and contribute to the regulation of older, conserved biological functions. One such gene, the novel insect-specific gene oskar, was first identified based on its role in establishing the Drosophila melanogaster germ line. We previously showed that this gene likely arose through an unusual domain transfer event involving bacterial endosymbionts and played a somatic role before evolving its well-known germ line function. Here, we provide empirical support for this hypothesis in the form of evidence for a neural role for oskar. We show that oskar is expressed in the adult neural stem cells of a hemimetabolous insect, the cricket Gryllus bimaculatus. In these stem cells, called neuroblasts, oskar is required together with the ancient animal transcription factor Creb to regulate long-term (but not short-term) olfactory memory. We provide evidence that oskar positively regulates Creb, which plays a conserved role in long-term memory across animals, and that oskar in turn may be a direct target of Creb. Together with previous reports of a role for oskar in nervous system development and function in crickets and flies, our results are consistent with the hypothesis that oskar's original somatic role may have been in the insect nervous system. Moreover, its colocalization and functional cooperation with the conserved pluripotency gene piwi in the nervous system may have facilitated oskar's later co-option to the germ line in holometabolous insects.

Emergence of an Orphan Nitrogenase Protein Following Atmospheric Oxygenation.

Molecular innovations within key metabolisms can have profound impacts on element cycling and ecological distribution. Yet, much of the molecular foundations of early evolved enzymes and metabolisms are unknown. Here, we bring one such mystery to relief by probing the birth and evolution of the G-subunit protein, an integral component of certain members of the nitrogenase family, the only enzymes capable of biological nitrogen fixation. The G-subunit is a Paleoproterozoic-age orphan protein that appears more than 1 billion years after the origin of nitrogenases. We show that the G-subunit arose with novel nitrogenase metal dependence and the ecological expansion of nitrogen-fixing microbes following the transition in environmental metal availabilities and atmospheric oxygenation that began ∼2.5 billion years ago. We identify molecular features that suggest early G-subunit proteins mediated cofactor or protein interactions required for novel metal dependency, priming ancient nitrogenases and their hosts to exploit these newly diversified geochemical environments. We further examined the degree of functional specialization in G-subunit evolution with extant and ancestral homologs using laboratory reconstruction experiments. Our results indicate that permanent recruitment of the orphan protein depended on the prior establishment of conserved molecular features and showcase how contingent evolutionary novelties might shape ecologically important microbial innovations.

Phylogenomics provides insights into the evolution of cactophily and host plant shifts in Drosophila.

Cactophilic species of the Drosophila buzzatii cluster (repleta group) comprise an excellent model group to investigate genomic changes underlying adaptation to extreme climate conditions and host plants. In particular, these species form a tractable system to study the transition from chemically simpler breeding sites (like prickly pears of the genus Opuntia) to chemically more complex hosts (columnar cacti). Here, we report four highly contiguous genome assemblies of three species of the buzzatii cluster. Based on this genomic data and inferred phylogenetic relationships, we identified candidate taxonomically restricted genes (TRGs) likely involved in the evolution of cactophily and cactus host specialization. Functional enrichment analyses of TRGs within the buzzatii cluster identified genes involved in detoxification, water preservation, immune system response, anatomical structure development, and morphogenesis. In contrast, processes that regulate responses to stress, as well as the metabolism of nitrogen compounds, transport, and secretion were found in the set of species that are columnar cacti dwellers. These findings are in line with the hypothesis that those genomic changes brought about key mechanisms underlying the adaptation of the buzzatii cluster species to arid regions in South America.

Transcriptional and post-transcriptional regulation of young genes in plants.

BACKGROUND: New genes continuously emerge from non-coding DNA or by diverging from existing genes, but most of them are rapidly lost and only a few become fixed within the population. We hypothesized that young genes are subject to transcriptional and post-transcriptional regulation to limit their expression and minimize their exposure to purifying selection. RESULTS: We performed a protein-based homology search across the tree of life to determine the evolutionary age of protein-coding genes present in the rice genome. We found that young genes in rice have relatively low expression levels, which can be attributed to distal enhancers, and closed chromatin conformation at their transcription start sites (TSS). The chromatin in TSS regions can be re-modeled in response to abiotic stress, indicating conditional expression of young genes. Furthermore, transcripts of young genes in Arabidopsis tend to be targeted by nonsense-mediated RNA decay, presenting another layer of regulation limiting their expression. CONCLUSIONS: These data suggest that transcriptional and post-transcriptional mechanisms contribute to the conditional expression of young genes, which may alleviate purging selection while providing an opportunity for phenotypic exposure and functionalization.

Pleomorphic Variants of Borreliella (syn. Borrelia) burgdorferi Express Evolutionary Distinct Transcriptomes.

Borreliella (syn. Borrelia) burgdorferi is a spirochete bacterium that causes tick-borne Lyme disease. Along its lifecycle B. burgdorferi develops several pleomorphic forms with unclear biological and medical relevance. Surprisingly, these morphotypes have never been compared at the global transcriptome level. To fill this void, we grew B. burgdorferi spirochete, round body, bleb, and biofilm-dominated cultures and recovered their transcriptomes by RNAseq profiling. We found that round bodies share similar expression profiles with spirochetes, despite their morphological differences. This sharply contrasts to blebs and biofilms that showed unique transcriptomes, profoundly distinct from spirochetes and round bodies. To better characterize differentially expressed genes in non-spirochete morphotypes, we performed functional, positional, and evolutionary enrichment analyses. Our results suggest that spirochete to round body transition relies on the delicate regulation of a relatively small number of highly conserved genes, which are located on the main chromosome and involved in translation. In contrast, spirochete to bleb or biofilm transition includes substantial reshaping of transcription profiles towards plasmids-residing and evolutionary young genes, which originated in the ancestor of Borreliaceae. Despite their abundance the function of these Borreliaceae-specific genes is largely unknown. However, many known Lyme disease virulence genes implicated in immune evasion and tissue adhesion originated in this evolutionary period. Taken together, these regularities point to the possibility that bleb and biofilm morphotypes might be important in the dissemination and persistence of B. burgdorferi inside the mammalian host. On the other hand, they prioritize the large pool of unstudied Borreliaceae-specific genes for functional characterization because this subset likely contains undiscovered Lyme disease pathogenesis genes.

A deep learning approach for orphan gene identification in moso bamboo (Phyllostachys edulis) based on the CNN + Transformer model.

BACKGROUND: Orphan gene play an important role in the environmental stresses of many species and their identification is a critical step to understand biological functions. Moso bamboo has high ecological, economic and cultural value. Studies have shown that the growth of moso bamboo is influenced by various stresses. Several traditional methods are time-consuming and inefficient. Hence, the development of efficient and high-accuracy computational methods for predicting orphan genes is of great significance. RESULTS: In this paper, we propose a novel deep learning model (CNN + Transformer) for identifying orphan genes in moso bamboo. It uses a convolutional neural network in combination with a transformer neural network to capture k-mer amino acids and features between k-mer amino acids in protein sequences. The experimental results show that the average balance accuracy value of CNN + Transformer on moso bamboo dataset can reach 0.875, and the average Matthews Correlation Coefficient (MCC) value can reach 0.471. For the same testing set, the Balance Accuracy (BA), Geometric Mean (GM), Bookmaker Informedness (BM), and MCC values of the recurrent neural network, long short-term memory, gated recurrent unit, and transformer models are all lower than those of CNN + Transformer, which indicated that the model has the extensive ability for OG identification in moso bamboo. CONCLUSIONS: CNN + Transformer model is feasible and obtains the credible predictive results. It may also provide valuable references for other related research. As our knowledge, this is the first model to adopt the deep learning techniques for identifying orphan genes in plants.

Spatial Transcriptomics of Nematodes Identifies Sperm Cells as a Source of Genomic Novelty and Rapid Evolution.

Divergence of gene function and expression during development can give rise to phenotypic differences at the level of cells, tissues, organs, and ultimately whole organisms. To gain insights into the evolution of gene expression and novel genes at spatial resolution, we compared the spatially resolved transcriptomes of two distantly related nematodes, Caenorhabditis elegans and Pristionchus pacificus, that diverged 60-90 Ma. The spatial transcriptomes of adult worms show little evidence for strong conservation at the level of single genes. Instead, regional expression is largely driven by recent duplication and emergence of novel genes. Estimation of gene ages across anatomical structures revealed an enrichment of novel genes in sperm-related regions. This provides first evidence in nematodes for the "out of testis" hypothesis that has been previously postulated based on studies in Drosophila and mammals. "Out of testis" genes represent a mix of products of pervasive transcription as well as fast evolving members of ancient gene families. Strikingly, numerous novel genes have known functions during meiosis in Caenorhabditis elegans indicating that even universal processes such as meiosis may be targets of rapid evolution. Our study highlights the importance of novel genes in generating phenotypic diversity and explicitly characterizes gene origination in sperm-related regions. Furthermore, it proposes new functions for previously uncharacterized genes and establishes the spatial transcriptome of Pristionchus pacificus as a catalog for future studies on the evolution of gene expression and function.

New Genes Interacted With Recent Whole-Genome Duplicates in the Fast Stem Growth of Bamboos.

As drivers of evolutionary innovations, new genes allow organisms to explore new niches. However, clear examples of this process remain scarce. Bamboos, the unique grass lineage diversifying into the forest, have evolved with a key innovation of fast growth of woody stem, reaching up to 1 m/day. Here, we identify 1,622 bamboo-specific orphan genes that appeared in recent 46 million years, and 19 of them evolved from noncoding ancestral sequences with entire de novo origination process reconstructed. The new genes evolved gradually in exon-intron structure, protein length, expression specificity, and evolutionary constraint. These new genes, whether or not from de novo origination, are dominantly expressed in the rapidly developing shoots, and make transcriptomes of shoots the youngest among various bamboo tissues, rather than reproductive tissue in other plants. Additionally, the particularity of bamboo shoots has also been shaped by recent whole-genome duplicates (WGDs), which evolved divergent expression patterns from ancestral states. New genes and WGDs have been evolutionarily recruited into coexpression networks to underline fast-growing trait of bamboo shoot. Our study highlights the importance of interactions between new genes and genome duplicates in generating morphological innovation.

New Gene Origin and Deep Taxon Phylogenomics: Opportunities and Challenges.

The immense morphological and phenotypic diversity within eukaryotes coincides with large-scale differences in genic repertoires, including the presence of thousands of new genes in every genome. New genes arise through duplication and divergence of existing coding sequences or de novo from noncoding sequences. These processes together cause individual genomes to contain up to one-third of orphan genes without any detectable homology in other lineages. Recently, deep taxon phylogenomics, the genome comparisons of extremely closely related species, provided novel insight into the evolutionary dynamics of such rapidly evolving genes. This review focuses on deep taxon phylogenomics and its importance in studying the evolution of new genes and discusses challenges and opportunities.

Progress on plant orphan genes.

Orphan genes are located in a special evolutionary branch and have no significant sequence similarity with any other identified genes. Orphan genes are prevalent in every species, comparative genomics analyses found that all sequenced species contained a portion of orphan genes, and the number of orphan genes obtained by distinct screening conditions is different. Orphan genes are often associated with various stress responses, species-specific evolution and substance metabolism regulation. However, most of the orphan genes have not been well annotated or even have no recognizable functional domains, which brings some difficulties to the functional characterization of orphan genes. Compared with conserved genes, there is less research on orphan genes, which leads to the possibility that the importance of orphan genes may be "unrewarded". In this review, we summarize the origin and evolution of orphan genes, plant orphan gene screening and functions, and analyse the existing challenges and future research priorities and solutions, which provide theoretical basis for the study of orphan gene function and action mechanisms.

Transcriptional profiling reveals conserved and species-specific plant defense responses during the interaction of Physcomitrium patens with Botrytis cinerea.

KEY MESSAGE: Evolutionary conserved defense mechanisms present in extant bryophytes and angiosperms, as well as moss-specific defenses are part of the immune response of Physcomitrium patens. Bryophytes and tracheophytes are descendants of early land plants that evolved adaptation mechanisms to cope with different kinds of terrestrial stresses, including drought, variations in temperature and UV radiation, as well as defense mechanisms against microorganisms present in the air and soil. Although great advances have been made on pathogen perception and subsequent defense activation in angiosperms, limited information is available in bryophytes. In this study, a transcriptomic approach uncovered the molecular mechanisms underlying the defense response of the bryophyte Physcomitrium patens (previously Physcomitrella patens) against the important plant pathogen Botrytis cinerea. A total of 3.072 differentially expressed genes were significantly affected during B. cinerea infection, including genes encoding proteins with known functions in angiosperm immunity and involved in pathogen perception, signaling, transcription, hormonal signaling, metabolic pathways such as shikimate and phenylpropanoid, and proteins with diverse role in defense against biotic stress. Similarly as in other plants, B. cinerea infection leads to downregulation of genes involved in photosynthesis and cell cycle progression. These results highlight the existence of evolutionary conserved defense responses to pathogens throughout the green plant lineage, suggesting that they were probably present in the common ancestors of land plants. Moreover, several genes acquired by horizontal transfer from prokaryotes and fungi, and a high number of P. patens-specific orphan genes were differentially expressed during B. cinerea infection, suggesting that they are important players in the moss immune response.

Comparative genomics and community curation further improve gene annotations in the nematode Pristionchus pacificus.

BACKGROUND: Nematode model organisms such as Caenorhabditis elegans and Pristionchus pacificus are powerful systems for studying the evolution of gene function at a mechanistic level. However, the identification of P. pacificus orthologs of candidate genes known from C. elegans is complicated by the discrepancy in the quality of gene annotations, a common problem in nematode and invertebrate genomics. RESULTS: Here, we combine comparative genomic screens for suspicious gene models with community-based curation to further improve the quality of gene annotations in P. pacificus. We extend previous curations of one-to-one orthologs to larger gene families and also orphan genes. Cross-species comparisons of protein lengths, screens for atypical domain combinations and species-specific orphan genes resulted in 4311 candidate genes that were subject to community-based curation. Corrections for 2946 gene models were implemented in a new version of the P. pacificus gene annotations. The new set of gene annotations contains 28,896 genes and has a single copy ortholog completeness level of 97.6%. CONCLUSIONS: Our work demonstrates the effectiveness of comparative genomic screens to identify suspicious gene models and the scalability of community-based approaches to improve the quality of thousands of gene models. Similar community-based approaches can help to improve the quality of gene annotations in other invertebrate species, including parasitic nematodes.

Genome compartmentalization predates species divergence in the plant pathogen genus Zymoseptoria.

BACKGROUND: Antagonistic co-evolution can drive rapid adaptation in pathogens and shape genome architecture. Comparative genome analyses of several fungal pathogens revealed highly variable genomes, for many species characterized by specific repeat-rich genome compartments with exceptionally high sequence variability. Dynamic genome structure may enable fast adaptation to host genetics. The wheat pathogen Zymoseptoria tritici with its highly variable genome, has emerged as a model organism to study genome evolution of plant pathogens. Here, we compared genomes of Z. tritici isolates and of sister species infecting wild grasses to address the evolution of genome composition and structure. RESULTS: Using long-read technology, we sequenced and assembled genomes of Z. ardabiliae, Z. brevis, Z. pseudotritici and Z. passerinii, together with two isolates of Z. tritici. We report a high extent of genome collinearity among Zymoseptoria species and high conservation of genomic, transcriptomic and epigenomic signatures of compartmentalization. We identify high gene content variability both within and between species. In addition, such variability is mainly limited to the accessory chromosomes and accessory compartments. Despite strong host specificity and non-overlapping host-range between species, predicted effectors are mainly shared among Zymoseptoria species, yet exhibiting a high level of presence-absence polymorphism within Z. tritici. Using in planta transcriptomic data from Z. tritici, we suggest different roles for the shared orthologs and for the accessory genes during infection of their hosts. CONCLUSION: Despite previous reports of high genomic plasticity in Z. tritici, we describe here a high level of conservation in genomic, epigenomic and transcriptomic composition and structure across the genus Zymoseptoria. The compartmentalized genome allows the maintenance of a functional core genome co-occurring with a highly variable accessory genome.

Differences in gene expression in whitefly associated with CYSDV-infected and virus-free melon, and comparison with expression in whiteflies fed on ToCV- and TYLCV-infected tomato.

BACKGROUND: Cucurbit yellow stunting disorder virus (CYSDV; genus Crinivirus, Closteroviridae) is transmitted in a semipersistent manner by the whitefly, Bemisia tabaci, and is efficiently transmitted by the widely prevalent B. tabaci cryptic species, MEAM1. In this study, we compared transcriptome profiles of B. tabaci MEAM1, after 24 h, 72 h and 7 days of acquisition feeding on melon plants infected with CYSDV (CYSDV-whiteflies) with those fed on virus-free melon, using RNA-Seq technology. We also compared transcriptome profiles with whiteflies fed on tomato plants separately infected with Tomato chlorosis virus (ToCV), a crinivirus closely related to CYSDV, and Tomato yellow leaf curl virus (TYLCV), a member of the genus Begomovirus, which has a distinctly different mode of transmission and their respective virus-free controls, to find common gene expression changes among viruliferous whiteflies feeding on different host plants infected with distinct (TYLCV) and related (CYSDV and ToCV) viruses. RESULTS: A total of 275 differentially expressed genes (DEGs) were identified in CYSDV-whiteflies, with 3 DEGs at 24 h, 221 DEGs at 72 h, and 51 DEGs at 7 days of virus acquisition. Changes in genes encoding orphan genes (54 genes), phosphatidylethanolamine-binding proteins (PEBP) (20 genes), and AAA-ATPase domain containing proteins (10 genes) were associated with the 72 h time point. Several more orphan genes (20 genes) were differentially expressed at 7 days. A total of 59 common DEGs were found between CYSDV-whiteflies and ToCV-whiteflies, which included 20 orphan genes and 6 lysosomal genes. A comparison of DEGs across the three different virus-host systems revealed 14 common DEGs, among which, eight showed similar and significant up-regulation in CYSDV-whiteflies at 72 h and TYLCV-whiteflies at 24 h, while down-regulation of the same genes was observed in ToCV-whiteflies at 72 h. CONCLUSIONS: Dynamic gene expression changes occurred in CYSDV-whiteflies after 72 h feeding, with decreased gene expression changes associated with 7 days of CYSDV acquisition. Similarities in gene expression changes among CYSDV-whiteflies, ToCV-whiteflies and TYLCV-whiteflies suggest the possible involvement of common genes or pathways for virus acquisition and transmission by whiteflies, even for viruses with distinctly different modes of transmission.

De Novo Gene Evolution of Antifreeze Glycoproteins in Codfishes Revealed by Whole Genome Sequence Data.

New genes can arise through duplication of a pre-existing gene or de novo from non-coding DNA, providing raw material for evolution of new functions in response to a changing environment. A prime example is the independent evolution of antifreeze glycoprotein genes (afgps) in the Arctic codfishes and Antarctic notothenioids to prevent freezing. However, the highly repetitive nature of these genes complicates studies of their organization. In notothenioids, afgps evolved from an extant gene, yet the evolutionary origin of afgps in codfishes is unknown. Here, we demonstrate that afgps in codfishes have evolved de novo from non-coding DNA 13-18 Ma, coinciding with the cooling of the Northern Hemisphere. Using whole-genome sequence data from several codfishes and notothenioids, we find higher copy number of afgp in species exposed to more severe freezing suggesting a gene dosage effect. Notably, antifreeze function is lost in one lineage of codfishes analogous to the afgp losses in non-Antarctic notothenioids. This indicates that selection can eliminate the antifreeze function when freezing is no longer imminent. In addition, we show that evolution of afgp-assisting antifreeze potentiating protein genes (afpps) in notothenioids coincides with origin and lineage-specific losses of afgp. The origin of afgps in codfishes is one of the first examples of an essential gene born from non-coding DNA in a non-model species. Our study underlines the power of comparative genomics to uncover past molecular signatures of genome evolution, and further highlights the impact of de novo gene origin in response to a changing selection regime.

A morphological novelty evolved by co-option of a reduced gene regulatory network and gene recruitment in a beetle.

The mechanisms underlying the evolution of morphological novelties have remained enigmatic but co-option of existing gene regulatory networks (GRNs), recruitment of genes and the evolution of orphan genes have all been suggested to contribute. Here, we study a morphological novelty of beetle pupae called gin-trap. By combining the classical candidate gene approach with unbiased screening in the beetle Tribolium castaneum, we find that 70% of the tested components of the wing network were required for gin-trap development. However, many downstream and even upstream components were not included in the co-opted network. Only one gene was recruited from another biological context, but it was essential for the anteroposterior symmetry of the gin-traps, which represents a gin-trap-unique morphological innovation. Our data highlight the importance of co-option and modification of GRNs. The recruitment of single genes may not be frequent in the evolution of morphological novelties, but may be essential for subsequent diversification of the novelties. Finally, after having screened about 28% of annotated genes in the Tribolium genome to identify the genes required for gin-trap development, we found none of them are orphan genes, suggesting that orphan genes may have played only a minor, if any, role in the evolution of gin-traps.

Transcriptome analysis of the whitefly, Bemisia tabaci MEAM1 during feeding on tomato infected with the crinivirus, Tomato chlorosis virus, identifies a temporal shift in gene expression and differential regulation of novel orphan genes.

BACKGROUND: Whiteflies threaten agricultural crop production worldwide, are polyphagous in nature, and transmit hundreds of plant viruses. Little is known how whitefly gene expression is altered due to feeding on plants infected with a semipersistently transmitted virus. Tomato chlorosis virus (ToCV; genus Crinivirus, family Closteroviridae) is transmitted by the whitefly (Bemisia tabaci) in a semipersistent manner and infects several globally important agricultural and ornamental crops, including tomato. RESULTS: To determine changes in global gene regulation in whiteflies after feeding on tomato plants infected with a crinivirus (ToCV), comparative transcriptomic analysis was performed using RNA-Seq on whitefly (Bemisia tabaci MEAM1) populations after 24, 48, and 72 h acquisition access periods on either ToCV-infected or uninfected tomatoes. Significant differences in gene expression were detected between whiteflies fed on ToCV-infected tomato and those fed on uninfected tomato among the three feeding time periods: 447 up-regulated and 542 down-regulated at 24 h, 4 up-regulated and 7 down-regulated at 48 h, and 50 up-regulated and 160 down-regulated at 72 h. Analysis revealed differential regulation of genes associated with metabolic pathways, signal transduction, transport and catabolism, receptors, glucose transporters, α-glucosidases, and the uric acid pathway in whiteflies fed on ToCV-infected tomatoes, as well as an abundance of differentially regulated novel orphan genes. Results demonstrate for the first time, a specific and temporally regulated response by the whitefly to feeding on a host plant infected with a semipersistently transmitted virus, and advance the understanding of the whitefly vector-virus interactions that facilitate virus transmission. CONCLUSION: Whitefly transmission of semipersistent viruses is believed to require specific interactions between the virus and its vector that allow binding of virus particles to factors within whitefly mouthparts. Results provide a broader understanding of the potential mechanism of crinivirus transmission by whitefly, aid in discerning genes or loci in whitefly that influence virus interactions or transmission, and subsequently facilitate development of novel, genetics-based control methods against whitefly and whitefly-transmitted viruses.

Rethinking the role of immunity: lessons from Hydra.

The ability of multicellular organisms to detect and respond to microorganisms is fundamental and has ancient evolutionary origins. In this review, I evaluate our current understanding of the evolution of epithelial-based innate immunity in Hydra, an apparently simple animal that shares deep evolutionary connections with all animals, including humans. I highlight growing evidence that the innate immune system with its host-specific antimicrobial peptides and rich repertoire of pattern recognition receptors has evolved in response to the need for controlling resident beneficial microbes rather than to defend against invasive pathogens. These findings provide new insight into how developmental pathways beyond those associated with the immune system, such as stem cell transcriptional programs, interact with environmental cues such as microbes.

Are orphan genes protein-coding, prediction artifacts, or non-coding RNAs?

BACKGROUND: Current genome sequencing projects reveal substantial numbers of taxonomically restricted, so called orphan genes that lack homology with genes from other evolutionary lineages. However, it is not clear to what extent orphan genes are real, genomic artifacts, or represent non-coding RNAs. RESULTS: Here, we use a simple set of assumptions to test the nature of orphan genes. First, a sequence that is transcribed is considered a real biological entity. Second, every sequence that is supported by proteome data or shows a depletion of non-synonymous substitutions is a protein-coding gene. Using genomic, transcriptomic and proteomic data for the nematode Pristionchus pacificus, we show that between 4129-7997 (42-81 %) of predicted orphan genes are expressed and 3818-7545 (39-76 %) of orphan genes are under negative selection. In three cases that exhibited strong evolutionary constraint but lacked expression evidence in 14 RNA-seq samples, we could experimentally validate the predicted gene structures. Comparing different data sets to infer selection on orphan gene clusters, we find that the presence of a closely related genome provides the most powerful resource to robustly identify evidence of negative selection. However, even in the absence of other genomic data, the availability of paralogous sequences was enough to show negative selection in 8-10 % of orphan genes. CONCLUSIONS: Our study shows that the great majority of previously identified orphan genes in P. pacificus are indeed protein-coding genes. Even though this work represents a case study on a single species, our approach can be transferred to genomic data of other non-model organisms in order to ascertain the protein-coding nature of orphan genes.

Implementation and assessment of a yeast orphan gene research project: involving undergraduates in authentic research experiences and progressing our understanding of uncharacterized open reading frames.

Saccharomyces cerevisiae was the first eukaryotic organism to be sequenced; however, little progress has been made in recent years in furthering our understanding of all open reading frames (ORFs). From October 2012 to May 2015 the number of verified ORFs had only risen from 75.31% to 78%, while the number of uncharacterized ORFs had decreased from 12.8% to 11% (representing > 700 genes still left in this category; http://www.yeastgenome.org/genomesnapshot). Course-based research has been shown to increase student learning while providing experience with real scientific investigation; however, implementation in large, multi-section courses presents many challenges. This study sought to test the feasibility and effectiveness of incorporating authentic research into a core genetics course, with multiple instructors, to increase student learning and progress our understanding of uncharacterized ORFs. We generated a module-based annotation toolkit and utilized easily accessible bioinformatics tools to predict gene function for uncharacterized ORFs within the Saccharomyces Genome Database (SGD). Students were each assigned an uncharacterized ORF, which they annotated using contemporary comparative genomics methodologies, including multiple sequence alignment, conserved domain identification, signal peptide prediction and cellular localization algorithms. Student learning outcomes were measured by quizzes, project reports and presentations, as well as a post-project questionnaire. Our results indicate that the authentic research experience had positive impacts on students' perception of their learning and their confidence to conduct future research. Furthermore, we believe that creation of an online repository and adoption and/or adaptation of this project across multiple researchers and institutions could speed the process of gene function prediction.