To connect human biology to fish biomedical models, we sequenced the genome of spotted gar (Lepisosteus oculatus), whose lineage diverged from teleosts before the teleost genome duplication (TGD). The slowly evolving gar genome conserved in content and size many entire chromosomes from bony vertebrate ancestors. Gar bridges teleosts to tetrapods by illuminating the evolution of immunity, mineralization, and development (e.g., Hox, ParaHox, and miRNA genes). Numerous conserved non-coding elements (CNEs, often cis-regulatory) undetectable in direct human-teleost comparisons become apparent using gar: functional studies uncovered conserved roles of such cryptic CNEs, facilitating annotation of sequences identified in human genome-wide association studies. Transcriptomic analyses revealed that the sum of expression domains and levels from duplicated teleost genes often approximate patterns and levels of gar genes, consistent with subfunctionalization. The gar genome provides a resource for understanding evolution after genome duplication, the origin of vertebrate genomes, and the function of human regulatory sequences.

Sex determination can be robustly genetic, strongly environmental, or genetic subject to environmental perturbation. The genetic basis of sex determination is unknown for zebrafish (Danio rerio), a model for development and human health. We used RADtag population genomics to identify sex-linked polymorphisms. After verifying this "RAD-sex" method on medaka (Oryzias latipes), we studied two domesticated zebrafish strains (AB and TU), two natural laboratory strains (WIK and EKW), and two recent isolates from nature (NA and CB). All four natural strains had a single sex-linked region at the right tip of chromosome 4, enabling sex genotyping by PCR. Genotypes for the single nucleotide polymorphism (SNP) with the strongest statistical association to sex suggested that wild zebrafish have WZ/ZZ sex chromosomes. In natural strains, "male genotypes" became males and some "female genotypes" also became males, suggesting that the environment or genetic background can cause female-to-male sex reversal. Surprisingly, TU and AB lacked detectable sex-linked loci. Phylogenomics rooted on D. nigrofasciatus verified that all strains are monophyletic. Because AB and TU branched as a monophyletic clade, we could not rule out shared loss of the wild sex locus in a common ancestor despite their independent domestication. Mitochondrial DNA sequences showed that investigated strains represent only one of the three identified zebrafish haplogroups. Results suggest that zebrafish in nature possess a WZ/ZZ sex-determination mechanism with a major determinant lying near the right telomere of chromosome 4 that was modified during domestication. Strains providing the zebrafish reference genome lack key components of the natural sex-determination system but may have evolved variant sex-determining mechanisms during two decades in laboratory culture.. . .researchers using standard lines of zebrafish that have long been maintained in laboratories are often plagued by severe sex ratio distortions. . .. C. Lawrence, J. P. Ebersole, and R. V. Kesseli (2008) C ONSIDERING the fundamental importance of sex for species propagation, it is surprising that primary sexdetermining mechanisms are not strongly conserved among animal taxa (Bull 1983;Charlesworth 1996;Ming et al. 2011;Bachtrog et al. 2014). Closely related species or even populations of the same species can have different sexdetermining mechanisms (Takehana et al. 2007;Ross et al. 2009;Kobayashi et al. 2013;Heule et al. 2014;Larney et al. 2014). Zebrafish (Danio rerio) is a popular model for studies of vertebrate development, behavior, physiology, evolution, disease, and human health (Mills et al. 2007;Seth et al. 2013;Braasch et al. 2014;Ota and Kawahara 2014;Wilkinson et al. 2014), but researchers struggle with highly variable and distorted sex ratios, and investigations into the genetic nature of zebrafish sex determination are conflicting. To help understand these issues, we conducted a population genomic study of sex determination in multiple zebrafish strains. Zebrafish exhibit...

Within vertebrates, major sex determining genes can differ among taxa and even within species. In zebrafish (Danio rerio), neither heteromorphic sex chromosomes nor single sex determination genes of large effect, like Sry in mammals, have yet been identified. Furthermore, environmental factors can influence zebrafish sex determination. Although progress has been made in understanding zebrafish gonad differentiation (e.g. the influence of germ cells on gonad fate), the primary genetic basis of zebrafish sex determination remains poorly understood. To identify genetic loci associated with sex, we analyzed F2 offspring of reciprocal crosses between Oregon *AB and Nadia (NA) wild-type zebrafish stocks. Genome-wide linkage analysis, using more than 5,000 sequence-based polymorphic restriction site associated (RAD-tag) markers and population genomic analysis of more than 30,000 single nucleotide polymorphisms in our *ABxNA crosses revealed a sex-associated locus on the end of the long arm of chr-4 for both cross families, and an additional locus in the middle of chr-3 in one cross family. Additional sequencing showed that two SNPs in dmrt1 previously suggested to be functional candidates for sex determination in a cross of ABxIndia wild-type zebrafish, are not associated with sex in our AB fish. Our data show that sex determination in zebrafish is polygenic and that different genes may influence sex determination in different strains or that different genes become more important under different environmental conditions. The association of the end of chr-4 with sex is remarkable because, unique in the karyotype, this chromosome arm shares features with known sex chromosomes: it is highly heterochromatic, repetitive, late replicating, and has reduced recombination. Our results reveal that chr-4 has functional and structural properties expected of a sex chromosome.

Icefishes (suborder Notothenioidei; family Channichthyidae) are the only vertebrates that lack functional haemoglobin genes and red blood cells. Here, we report a high-quality genome assembly and linkage map for the Antarctic blackfin icefish Chaenocephalus aceratus, highlighting evolved genomic features for its unique physiology. Phylogenomic analysis revealed that Antarctic fish of the teleost suborder Notothenioidei, including icefishes, diverged from the stickleback lineage about 77 million years ago and subsequently evolved cold-adapted phenotypes as the Southern Ocean cooled to sub-zero temperatures. Our results show that genes involved in protection from ice damage, including genes encoding antifreeze glycoprotein and zona pellucida proteins, are highly expanded in the icefish genome. Furthermore, genes that encode enzymes that help to control cellular redox state, including members of the sod3 and nqo1 gene families, are expanded, probably as evolutionary adaptations to the relatively high concentration of oxygen dissolved in cold Antarctic waters. In contrast, some crucial regulators of circadian homeostasis (cry and per genes) are absent from the icefish genome, suggesting compromised control of biological rhythms in the polar light environment. The availability of the icefish genome sequence will accelerate our understanding of adaptation to extreme Antarctic environments.

High-throughput sequencing of microRNAs has revealed the diversity and variability of mature and functional short non-coding RNAs, including their genomic origins, biogenesis pathways, sequence variability, and newly identified products, such as microRNA-offset RNAs. Here, we review known cases of alternative mature miRNA-like RNA fragments and propose a revised definition of microRNAs to encompass this diversity. We then review nomenclature guidelines for microRNAs and propose to extend nomenclature conventions to align with those for protein-coding genes established by international consortia. Finally, we suggest a system to encompass the full complexity of sequence variations, i.e. isomiRs, in the analysis of small RNA sequencing experiments.

Many fields of biology – including vertebrate Evo-Devo research – are facing an explosion of genomic and transcriptomic sequence information and a multitude of fish species are now swimming in this ‘genomic tsunami’. Here, we first give an overview of recent developments in sequencing fish genomes and transcriptomes that identify properties of fish genomes requiring particular attention and propose strategies to overcome common challenges in fish genomics. We suggest that the generation of chromosome-level genome assemblies - for which we introduce the term ‘chromonome’ – should be a key component of genomic investigations in fish because they enable large-scale conserved synteny analyses that inform orthology detection, a process critical for connectivity of genomes. Orthology calls in vertebrates, especially in teleost fish, are complicated by divergent evolution of gene repertoires and functions following two rounds of genome duplication in the ancestor of vertebrates and a third round at the base of teleost fish. Second, using examples of spotted gar, basal teleosts, zebrafish-related cyprinids, cavefish, livebearers, icefish, and lobefin fish, we illustrate how next generation sequencing technologies liberate emerging fish systems from genomic ignorance and transform them into a new model army to answer longstanding questions on the genomic and developmental basis of their biodiversity. Finally, we discuss recent progress in the genetic toolbox for the major fish models for functional analysis, zebrafish and medaka, that can be transferred to many other fish species to study in vivo the functional effect of evolutionary genomic change as Evo-Devo research enters the postgenomic era.

Gar is an actinopterygian that has bone, dentin, enameloid, and ganoin (enamel) in teeth and/or scales. Mineralization of these tissues involves genes encoding various secretory calcium-binding phosphoproteins (SCPPs) in osteichthyans, but no SCPP genes have been identified in chondrichthyans to date. In the gar genome, we identified 38 SCPP genes, seven of which encode "acidic-residue-rich" proteins and 31 encode "Pro/Gln (P/Q) rich" proteins. These gar SCPP genes constitute the largest known repertoire, including many newly identified P/Q-rich genes expressed in teeth and/or scales. Among gar SCPP genes, six acidic and three P/Q-rich genes were identified as orthologs of sarcopterygian genes. The sarcopterygian orthologs of most of these acidic genes are involved in bone and/or dentin formation, and sarcopterygian orthologs of all three P/Q-rich genes participate in enamel formation. The finding of these genes in gar suggests that an elaborate SCPP gene-based genetic system for tissue mineralization was already present in stem osteichthyans. While SCPP genes have been thought to originate from ancient SPARCL1, SPARCL1L1 appears to be more closely related to these genes, because it established a structure similar to acidic SCPP genes probably in stem gnathostomes, perhaps at about the same time with the origin of tissue mineralization. Assuming enamel evolved in stem osteichthyans, all P/Q-rich SCPP genes likely arose within the osteichthyan lineage. Furthermore, the absence of acidic SCPP genes in chondrichthyans might be explained by the secondary loss of earliest acidic genes. It appears that many SCPP genes expanded rapidly in stem osteichthyans and in basal actinopterygians.

MicroRNAs (miRNAs) can have organ-specific expression and functions; they can originate from dedicated miRNA genes, from non-canonical miRNA genes, or from mirror-miRNA genes and can also experience post-transcriptional variation. It remains unclear, however, which mechanisms of miRNA production or modification are organ-specific and the extent of their evolutionary conservation. To address these issues, we developed the software Prost! (PRocessing Of Short Transcripts), which, among other features, helps quantify mature miRNAs, accounts for post-transcriptional processing, such as nucleotide editing, and identifies mirror-miRNAs. Here, we applied Prost! to annotate and analyze miRNAs in three-spined stickleback ( Gasterosteus aculeatus ), a model fish for evolutionary biology reported to have a miRNome larger than most teleost fish. Zebrafish ( Danio rerio ), a distantly related teleost with a well-known miRNome, served as comparator. Our results provided evidence for the existence of 286 miRNA genes and 382 unique mature miRNAs (excluding mir430 gene duplicates and the vaultRNA-derived mir733 ), which doesn’t represent a miRNAome larger than other teleost miRNomes. In addition, small RNA sequencing data from brain, heart, testis, and ovary in both stickleback and zebrafish identified suites of mature miRNAs that display organ-specific enrichment, many of which are evolutionarily-conserved in the brain and heart in both species. These data also supported the hypothesis that evolutionarily-conserved, organ-specific mechanisms may regulate post-transcriptional variations in miRNA sequence. In both stickleback and zebrafish, miR2188-5p was edited frequently with similar nucleotide changes in the seed sequence with organ specific editing rates, highest in the brain. In summary, Prost! is a new tool to identify and understand small RNAs, to help clarify a species’ miRNA biology as shown here for an important model for the evolution of developmental mechanisms, and to provide insight into organ-enriched expression and the evolutionary conservation of miRNA post-transcriptional modifications.