Understanding the mechanisms driving lineage-specific evolution in both primates and rodents has been hindered by the lack of sister clades with a similar phylogenetic structure having high-quality genome assemblies. Here, we have created chromosome-level assemblies of the Mus caroli and Mus pahari genomes. Together with the Mus musculus and Rattus norvegicus genomes, this set of rodent genomes is similar in divergence times to the Hominidae (human-chimpanzee-gorilla-orangutan). By comparing the evolutionary dynamics between the Muridae and Hominidae, we identified punctate events of chromosome reshuffling that shaped the ancestral karyotype of Mus musculus and Mus caroli between 3 and 6 million yr ago, but that are absent in the Hominidae. Hominidae show between four- and sevenfold lower rates of nucleotide change and feature turnover in both neutral and functional sequences, suggesting an underlying coherence to the Muridae acceleration. Our system of matched, high-quality genome assemblies revealed how specific classes of repeats can play lineage-specific roles in related species. Recent LINE activity has remodeled protein-coding loci to a greater extent across the Muridae than the Hominidae, with functional consequences at the species level such as reproductive isolation. Furthermore, we charted a Muridae-specific retrotransposon expansion at unprecedented resolution, revealing how a single nucleotide mutation transformed a specific SINE element into an active CTCF binding site carrier specifically in Mus caroli, which resulted in thousands of novel, species-specific CTCF binding sites. Our results show that the comparison of matched phylogenetic sets of genomes will be an increasingly powerful strategy for understanding mammalian biology.

For 10,000 years pigs and humans have shared a close and complex relationship. From domestication to modern breeding practices, humans have shaped the genomes of domestic pigs. Here we present the assembly and analysis of the genome sequence of a female domestic Duroc pig (Sus scrofa) and a comparison with the genomes of wild and domestic pigs from Europe and Asia. Wild pigs emerged in South East Asia and subsequently spread across Eurasia. Our results reveal a deep phylogenetic split between European and Asian wild boars ∼1 million years ago, and a selective sweep analysis indicates selection on genes involved in RNA processing and regulation. Genes associated with immune response and olfaction exhibit fast evolution. Pigs have the largest repertoire of functional olfactory receptor genes, reflecting the importance of smell in this scavenging animal. The pig genome sequence provides an important resource for further improvements of this important livestock species, and our identification of many putative disease-causing variants extends the potential of the pig as a biomedical model.

The Antarctic icefishes (family Channichthyidae, suborder Notothenioidei) constitute the only vertebrate taxon that fails to produce red blood cells. These fishes can be paired with closely related, but erythrocyte-producing, notothenioids to discover erythropoietic genes via representational difference analysis. Using a B30.2-domain-encoding DNA probe so derived from the hematopoietic kidney (pronephros) of a red-blooded Antarctic rockcod, Notothenia coriiceps, we discovered a related, novel gene, bloodthirsty (bty), that encoded a 547-residue protein that contains sequential RING finger, B Box, coiled-coil, and B30.2 domains. bty mRNA was expressed by the pronephric kidney of N. coriiceps at a steady-state level 10-fold greater than that found in the kidney of the icefish Chaenocephalus aceratus. To test the function of bty, we cloned the orthologous zebrafish gene from a kidney cDNA library. Whole-mount in situ hybridization of zebrafish embryos showed that bty mRNA was present throughout development and, after the mid-blastula transition, was expressed in the head and in or near the site of primitive erythropoiesis in the tail just prior to red cell production. One- to four-cell embryos injected with two distinct antisense morpholino oligonucleotides (MOs) targeted to the 5'-end of the bty mRNA failed to develop red cells, whereas embryos injected with 4- and 5-bp mismatch control MOs produced wild-type quantities of erythrocytes. The morphant phenotype was rescued by co-injection of synthetic bty mRNA containing an artificial 5'-untranslated region (UTR) with the antisense MO that bound the 5'-UTR of the wild-type bty transcript. Furthermore, the expression of genes that mark terminal erythroid differentiation was greatly reduced in the antisense-MO-treated embryos. We conclude that bty is likely to play a role in differentiation of the committed red cell progenitor.

Pedomorphism is the retention of ancestrally juvenile traits by adults in a descendant taxon. Despite its importance for evolutionary change, there are few examples of a molecular basis for this phenomenon. Notothenioids represent one of the best described species flocks among marine fishes, but their diversity is currently threatened by the rapidly changing Antarctic climate. Notothenioid evolutionary history is characterized by parallel radiations from a benthic ancestor to pelagic predators, which was accompanied by the appearance of several pedomorphic traits, including the reduction of skeletal mineralization that resulted in increased buoyancy.

The retina has a very high energy demand but lacks an internal blood supply in most vertebrates. Here we explore the hypothesis that oxygen diffusion limited the evolution of retinal morphology by reconstructing the evolution of retinal thickness and the various mechanisms for retinal oxygen supply, including capillarization and acid-induced haemoglobin oxygen unloading. We show that a common ancestor of bony fishes likely had a thin retina without additional retinal oxygen supply mechanisms and that three different types of retinal capillaries were gained and lost independently multiple times during the radiation of vertebrates, and that these were invariably associated with parallel changes in retinal thickness. Since retinal thickness confers multiple advantages to vision, we propose that insufficient retinal oxygen supply constrained the functional evolution of the eye in early vertebrates, and that recurrent origins of additional retinal oxygen supply mechanisms facilitated the phenotypic evolution of improved functional eye morphology.

In the frigid, oxygen-rich Southern Ocean (SO), Antarctic icefishes (Channichthyidae; Notothenioidei) evolved the ability to survive without producing erythrocytes and hemoglobin, the oxygen-transport system of virtually all vertebrates. Here, we integrate paleoclimate records with an extensive phylogenomic dataset of notothenioid fishes to understand the evolution of trait loss associated with climate change. In contrast to buoyancy adaptations in this clade, we find relaxed selection on the genetic regions controlling erythropoiesis evolved only after sustained cooling in the SO. This pattern is seen not only within icefishes but also occurred independently in other high-latitude notothenioids. We show that one species of the red-blooded dragonfish clade evolved a spherocytic anemia that phenocopies human patients with this disease via orthologous mutations. The genomic imprint of SO climate change is biased toward erythrocyte-associated conserved noncoding elements (CNEs) rather than to coding regions, which are largely preserved through pleiotropy. The drift in CNEs is specifically enriched near genes that are preferentially expressed late in erythropoiesis. Furthermore, we find that the hematopoietic marrow of icefish species retained proerythroblasts, which indicates that early erythroid development remains intact. Our results provide a framework for understanding the interactions between development and the genome in shaping the response of species to climate change.

The vaquita is the most critically endangered marine mammal, with fewer than 19 remaining in the wild. First described in 1958, the vaquita has been in rapid decline for more than 20 years resulting from inadvertent deaths due to the increasing use of large-mesh gillnets. To understand the evolutionary and demographic history of the vaquita, we used combined long-read sequencing and long-range scaffolding methods with long- and short-read RNA sequencing to generate a near error-free annotated reference genome assembly from cell lines derived from a female individual. The genome assembly consists of 99.92% of the assembled sequence contained in 21 nearly gapless chromosome-length autosome scaffolds and the X-chromosome scaffold, with a scaffold N50 of 115 Mb. Genome-wide heterozygosity is the lowest (0.01%) of any mammalian species analysed to date, but heterozygosity is evenly distributed across the chromosomes, consistent with long-term small population size at genetic equilibrium, rather than low diversity resulting from a recent population bottleneck or inbreeding. Historical demography of the vaquita indicates long-term population stability at less than 5,000 (Ne) for over 200,000 years. Together, these analyses indicate that the vaquita genome has had ample opportunity to purge highly deleterious alleles and potentially maintain diversity necessary for population health.

Modern sequencing technologies should make the assembly of the relatively small mitochondrial genomes an easy undertaking. However, few tools exist that address mitochondrial assembly directly.

The zebra finch is one of the most commonly studied songbirds in biology, particularly in genomics, neuroscience and vocal communication. However, this species lacks a robust cell line for molecular biology research and reagent optimization. We generated a cell line, designated CFS414, from zebra finch embryonic fibroblasts using the SV40 large and small T antigens. This cell line demonstrates an improvement over previous songbird cell lines through continuous and density-independent growth, allowing for indefinite culture and monoclonal line derivation. Cytogenetic, genomic, and transcriptomic profiling established the provenance of this cell line and identified the expression of genes relevant to ongoing songbird research. Using this cell line, we disrupted endogenous gene sequences using S.aureus Cas9 and confirmed a stress-dependent localization response of a song system specialized gene, SAP30L. The utility of CFS414 cells enhances the comprehensive molecular potential of the zebra finch and validates cell immortalization strategies in a songbird species.

The ring-tailed lemur (Lemur catta) is a charismatic strepsirrhine primate endemic to Madagascar. These lemurs are of particular interest, given their status as a flagship species and widespread publicity in the popular media. Unfortunately, a recent population decline has resulted in the census population decreasing to <2,500 individuals in the wild, and the species's classification as an endangered species by the IUCN. As is the case for most strepsirrhine primates, only a limited amount of genomic research has been conducted on L. catta, in part owing to the lack of genomic resources.

Accurate reference genome sequences provide the foundation for modern molecular biology and genomics as the interpretation of sequence data to study evolution, gene expression, and epigenetics depends heavily on the quality of the genome assembly used for its alignment. Correctly organising sequenced fragments such as contigs and scaffolds in relation to each other is a critical and often challenging step in the construction of robust genome references. We previously identified misoriented regions in the mouse and human reference assemblies using Strand-seq, a single cell sequencing technique that preserves DNA directionality Here we demonstrate the ability of Strand-seq to build and correct full-length chromosomes by identifying which scaffolds belong to the same chromosome and determining their correct order and orientation, without the need for overlapping sequences. We demonstrate that Strand-seq exquisitely maps assembly fragments into large related groups and chromosome-sized clusters without using new assembly data. Using template strand inheritance as a bi-allelic marker, we employ genetic mapping principles to cluster scaffolds that are derived from the same chromosome and order them within the chromosome based solely on directionality of DNA strand inheritance. We prove the utility of our approach by generating improved genome assemblies for several model organisms including the ferret, pig, Xenopus, zebrafish, Tasmanian devil and the Guinea pig.

Long COVID in children and adolescents remains poorly understood due to a lack of well-controlled studies with long-term follow-up. In particular, the impact of the family context on persistent symptoms following SARS-CoV-2 infection remains unknown. We examined long COVID symptoms in a cohort of infected children, adolescents, and adults and their exposed but non-infected household members approximately 1 year after infection and investigated clustering of persistent symptoms within households.

Sharks occupy diverse ecological niches and play critical roles in marine ecosystems, often acting as apex predators. They are considered a slow-evolving lineage and have been suggested to exhibit exceptionally low cancer rates. These two features could be explained by a low nuclear mutation rate. Here, we provide a direct estimate of the nuclear mutation rate in the epaulette shark (Hemiscyllium ocellatum). We generate a high-quality reference genome, and resequence the whole genomes of parents and nine offspring to detect de novo mutations. Using stringent criteria, we estimate a mutation rate of 7×10-10 per base pair, per generation. This represents one of the lowest directly estimated mutation rates for any vertebrate clade, indicating that this basal vertebrate group is indeed a slowly evolving lineage whose ability to restore genetic diversity following a sustained population bottleneck may be hampered by a low mutation rate.

The seventh iteration of the reference genome assembly for Rattus norvegicus-mRatBN7.2-corrects numerous misplaced segments and reduces base-level errors by approximately 9-fold and increases contiguity by 290-fold compared to its predecessor. Gene annotations are now more complete, significantly improving the mapping precision of genomic, transcriptomic, and proteomics data sets. We jointly analyzed 163 short-read whole genome sequencing datasets representing 120 laboratory rat strains and substrains using mRatBN7.2. We defined ~20.0 million sequence variations, of which 18.7 thousand are predicted to potentially impact the function of 6,677 genes. We also generated a new rat genetic map from 1,893 heterogeneous stock rats and annotated transcription start sites and alternative polyadenylation sites. The mRatBN7.2 assembly, along with the extensive analysis of genomic variations among rat strains, enhances our understanding of the rat genome, providing researchers with an expanded resource for studies involving rats.

Combining genome assembly with population and functional genomics can provide valuable insights to development and evolution, as well as tools for species management. Here, we present a chromosome-level genome assembly of the common brushtail possum (Trichosurus vulpecula), a model marsupial threatened in parts of their native range in Australia, but also a major introduced pest in New Zealand. Functional genomics reveals post-natal activation of chemosensory and metabolic genes, reflecting unique adaptations to altricial birth and delayed weaning, a hallmark of marsupial development. Nuclear and mitochondrial analyses trace New Zealand possums to distinct Australian subspecies, which have subsequently hybridised. This admixture allowed phasing of parental alleles genome-wide, ultimately revealing at least four genes with imprinted, parent-specific expression not yet detected in other species (MLH1, EPM2AIP1, UBP1 and GPX7). We find that reprogramming of possum germline imprints, and the wider epigenome, is similar to eutherian mammals except onset occurs after birth. Together, this work is useful for genetic-based control and conservation of possums, and contributes to understanding of the evolution of novel mammalian epigenetic traits.

We have generated an improved assembly and gene annotation of the pig X Chromosome, and a first draft assembly of the pig Y Chromosome, by sequencing BAC and fosmid clones from Duroc animals and incorporating information from optical mapping and fiber-FISH. The X Chromosome carries 1033 annotated genes, 690 of which are protein coding. Gene order closely matches that found in primates (including humans) and carnivores (including cats and dogs), which is inferred to be ancestral. Nevertheless, several protein-coding genes present on the human X Chromosome were absent from the pig, and 38 pig-specific X-chromosomal genes were annotated, 22 of which were olfactory receptors. The pig Y-specific Chromosome sequence generated here comprises 30 megabases (Mb). A 15-Mb subset of this sequence was assembled, revealing two clusters of male-specific low copy number genes, separated by an ampliconic region including the HSFY gene family, which together make up most of the short arm. Both clusters contain palindromes with high sequence identity, presumably maintained by gene conversion. Many of the ancestral X-related genes previously reported in at least one mammalian Y Chromosome are represented either as active genes or partial sequences. This sequencing project has allowed us to identify genes--both single copy and amplified--on the pig Y Chromosome, to compare the pig X and Y Chromosomes for homologous sequences, and thereby to reveal mechanisms underlying pig X and Y Chromosome evolution.

There are several protocols and kits for the extraction of circulating RNAs from plasma with a following quantification of specific genes via RT-qPCR. Due to the marginal amount of cell-free RNA in plasma samples, the total RNA yield is insufficient to perform Next-Generation Sequencing (NGS), the state-of-the-art technology in massive parallel sequencing that enables a comprehensive characterization of the whole transcriptome. Screening the transcriptome for biomarker signatures accelerates progress in biomarker profiling for molecular diagnostics, early disease detection or food safety. Therefore, the aim was to optimize a method that enables the extraction of sufficient amounts of total RNA from bovine plasma to generate good-quality small RNA Sequencing (small RNA-Seq) data. An increased volume of plasma (9 ml) was processed using the Qiagen miRNeasy Serum/Plasma Kit in combination with the QIAvac24 Plus system, a vacuum manifold that enables handling of high volumes during RNA isolation. 35 ng of total RNA were passed on to cDNA library preparation followed by small RNA high-throughput sequencing analysis on the Illumina HiSeq2000 platform. Raw sequencing reads were processed by a data analysis pipeline using different free software solutions. Seq-data was trimmed, quality checked, gradually selected for miRNAs/piRNAs and aligned to small RNA reference annotation indexes. Mapping to human reference indexes resulted in 4.8±2.8% of mature miRNAs and 1.4±0.8% of piRNAs and of 5.0±2.9% of mature miRNAs for bos taurus.

Antarctic fish have adapted to the freezing waters of the Southern Ocean. Representative adaptations to this harsh environment include a constitutive heat shock response and the evolution of an antifreeze protein in the blood. Despite their adaptations to the cold, genome-wide studies have not yet been performed on these fish due to the lack of a sequenced genome. Notothenia coriiceps, the Antarctic bullhead notothen, is an endemic teleost fish with a circumpolar distribution and makes a good model to understand the genomic adaptations to constant sub-zero temperatures.

Balanced chromosome abnormalities (BCAs) occur at a high frequency in healthy and diseased individuals, but cost-efficient strategies to identify BCAs and evaluate whether they contribute to a phenotype have not yet become widespread. Here we apply genome-wide mate-pair library sequencing to characterize structural variation in a patient with unclear neurodevelopmental disease (NDD) and complex de novo BCAs at the karyotype level. Nucleotide-level characterization of the clinically described BCA breakpoints revealed disruption of at least three NDD candidate genes (LINC00299, NUP205, PSMD14) that gave rise to abnormal mRNAs and could be assumed as disease-causing. However, unbiased genome-wide analysis of the sequencing data for cryptic structural variation was key to reveal an additional submicroscopic inversion that truncates the schizophrenia- and bipolar disorder-associated brain transcription factor ZNF804A as an equally likely NDD-driving gene. Deep sequencing of fluorescent-sorted wild-type and derivative chromosomes confirmed the clinically undetected BCA. Moreover, deep sequencing further validated a high accuracy of mate-pair library sequencing to detect structural variants larger than 10 kB, proposing that this approach is powerful for clinical-grade genome-wide structural variant detection. Our study supports previous evidence for a role of ZNF804A in NDD and highlights the need for a more comprehensive assessment of structural variation in karyotypically abnormal individuals and patients with neurocognitive disease to avoid diagnostic deception.

The multi-tubulin hypothesis proposes that each tubulin isotype performs a unique role, or subset of roles, in the universe of microtubule function(s). To test this hypothesis, we are investigating the functions of the recently discovered, noncanonical β-like tubulins (BLTs) of the ciliate, Tetrahymena thermophila. Tetrahymena forms 17 distinct microtubular structures whose assembly had been thought to be based on single α- and β-isotypes. However, completion of the macronuclear genome sequence of Tetrahymena demonstrated that this ciliate possessed a β-tubulin multigene family: two synonymous genes (BTU1 and BTU2) encode the canonical β-tubulin, BTU2, and six genes (BLT1-6) yield five divergent β-tubulin isotypes. In this report, we examine the structural features and functions of two of the BLTs (BLT1 and BLT4) and compare them to those of BTU2.