An atlas of regulatory elements in chicken: A resource for chicken genetics and genomics.

A comprehensive characterization of regulatory elements in the chicken genome across tissues will have substantial impacts on both fundamental and applied research. Here, we systematically identified and characterized regulatory elements in the chicken genome by integrating 377 genome-wide sequencing datasets from 23 adult tissues. In total, we annotated 1.57 million regulatory elements, representing 15 distinct chromatin states, and predicted about 1.2 million enhancer-gene pairs and 7662 super-enhancers. This functional annotation of the chicken genome should have wide utility on identifying regulatory elements accounting for gene regulation underlying domestication, selection, and complex trait regulation, which we explored. In short, this comprehensive atlas of regulatory elements provides the scientific community with a valuable resource for chicken genetics and genomics.

A genomic timescale for placental mammal evolution.

The precise pattern and timing of speciation events that gave rise to all living placental mammals remain controversial. We provide a comprehensive phylogenetic analysis of genetic variation across an alignment of 241 placental mammal genome assemblies, addressing prior concerns regarding limited genomic sampling across species. We compared neutral genome-wide phylogenomic signals using concatenation and coalescent-based approaches, interrogated phylogenetic variation across chromosomes, and analyzed extensive catalogs of structural variants. Interordinal relationships exhibit relatively low rates of phylogenomic conflict across diverse datasets and analytical methods. Conversely, X-chromosome versus autosome conflicts characterize multiple independent clades that radiated during the Cenozoic. Genomic time trees reveal an accumulation of cladogenic events before and immediately after the Cretaceous-Paleogene (K-Pg) boundary, implying important roles for Cretaceous continental vicariance and the K-Pg extinction in the placental radiation.

Evolution of the ancestral mammalian karyotype and syntenic regions.

Decrypting the rearrangements that drive mammalian chromosome evolution is critical to understanding the molecular bases of speciation, adaptation, and disease susceptibility. Using 8 scaffolded and 26 chromosome-scale genome assemblies representing 23/26 mammal orders, we computationally reconstructed ancestral karyotypes and syntenic relationships at 16 nodes along the mammalian phylogeny. Three different reference genomes (human, sloth, and cattle) representing phylogenetically distinct mammalian superorders were used to assess reference bias in the reconstructed ancestral karyotypes and to expand the number of clades with reconstructed genomes. The mammalian ancestor likely had 19 pairs of autosomes, with nine of the smallest chromosomes shared with the common ancestor of all amniotes (three still conserved in extant mammals), demonstrating a striking conservation of synteny for ∼320 My of vertebrate evolution. The numbers and types of chromosome rearrangements were classified for transitions between the ancestral mammalian karyotype, descendent ancestors, and extant species. For example, 94 inversions, 16 fissions, and 14 fusions that occurred over 53 My differentiated the therian from the descendent eutherian ancestor. The highest breakpoint rate was observed between the mammalian and therian ancestors (3.9 breakpoints/My). Reconstructed mammalian ancestor chromosomes were found to have distinct evolutionary histories reflected in their rates and types of rearrangements. The distributions of genes, repetitive elements, topologically associating domains, and actively transcribed regions in multispecies homologous synteny blocks and evolutionary breakpoint regions indicate that purifying selection acted over millions of years of vertebrate evolution to maintain syntenic relationships of developmentally important genes and regulatory landscapes of gene-dense chromosomes.

Augmin-dependent microtubule self-organization drives kinetochore fiber maturation in mammals.

Chromosome segregation in mammals relies on the maturation of a thick bundle of kinetochore-attached microtubules known as k-fiber. How k-fibers mature from initial kinetochore microtubule attachments remains a fundamental question. By combining molecular perturbations and phenotypic analyses in Indian muntjac fibroblasts containing the lowest known diploid chromosome number in mammals (2N = 6) and distinctively large kinetochores, with fixed/live-cell super-resolution coherent-hybrid stimulated emission depletion (CH-STED) nanoscopy and laser microsurgery, we demonstrate a key role for augmin in kinetochore microtubule self-organization and maturation, regardless of pioneer centrosomal microtubules. In doing so, augmin promotes kinetochore and interpolar microtubule turnover and poleward flux. Tracking of microtubule growth events within individual k-fibers reveals a wide angular dispersion, consistent with augmin-mediated branched microtubule nucleation. Augmin depletion reduces the frequency of kinetochore microtubule growth events and hampers efficient repair after acute k-fiber injury by laser microsurgery. Together, these findings underscore the contribution of augmin-mediated microtubule amplification for k-fiber self-organization and maturation in mammals.

Conservation of chromatin conformation in carnivores.

High throughput chromatin conformation capture (Hi-C) of leukocyte DNA was used to investigate the evolutionary stability of chromatin conformation at the chromosomal level in 11 species from three carnivore families: Felidae, Canidae, and Ursidae. Chromosome-scale scaffolds (C-scaffolds) of each species were initially used for whole-genome alignment to a reference genome within each family. This approach established putative orthologous relationships between C-scaffolds among the different species. Hi-C contact maps for all C-scaffolds were then visually compared and found to be distinct for a given reference chromosome or C-scaffold within a species and indistinguishable for orthologous C-scaffolds having a 1:1 relationship within a family. The visual patterns within families were strongly supported by eigenvectors from the Hi-C contact maps. Analysis of Hi-C contact maps and eigenvectors across the three carnivore families revealed that most cross-family orthologous subchromosomal fragments have a conserved three-dimensional (3D) chromatin structure and thus have been under strong evolutionary constraint for ∼54 My of carnivore evolution. The most pronounced differences in chromatin conformation were observed for the X chromosome and the red fox genome, whose chromosomes have undergone extensive rearrangements relative to other canids. We also demonstrate that Hi-C contact map pattern analysis can be used to accurately identify orthologous relationships between C-scaffolds and chromosomes, a method we termed "3D comparative scaffotyping." This method provides a powerful means for estimating karyotypes in de novo sequenced species that have unknown karyotype and no physical mapping information.

Why sequence all eukaryotes?

Life on Earth has evolved from initial simplicity to the astounding complexity we experience today. Bacteria and archaea have largely excelled in metabolic diversification, but eukaryotes additionally display abundant morphological innovation. How have these innovations come about and what constraints are there on the origins of novelty and the continuing maintenance of biodiversity on Earth? The history of life and the code for the working parts of cells and systems are written in the genome. The Earth BioGenome Project has proposed that the genomes of all extant, named eukaryotes-about 2 million species-should be sequenced to high quality to produce a digital library of life on Earth, beginning with strategic phylogenetic, ecological, and high-impact priorities. Here we discuss why we should sequence all eukaryotic species, not just a representative few scattered across the many branches of the tree of life. We suggest that many questions of evolutionary and ecological significance will only be addressable when whole-genome data representing divergences at all of the branchings in the tree of life or all species in natural ecosystems are available. We envisage that a genomic tree of life will foster understanding of the ongoing processes of speciation, adaptation, and organismal dependencies within entire ecosystems. These explorations will resolve long-standing problems in phylogenetics, evolution, ecology, conservation, agriculture, bioindustry, and medicine.

Standards recommendations for the Earth BioGenome Project.

A global international initiative, such as the Earth BioGenome Project (EBP), requires both agreement and coordination on standards to ensure that the collective effort generates rapid progress toward its goals. To this end, the EBP initiated five technical standards committees comprising volunteer members from the global genomics scientific community: Sample Collection and Processing, Sequencing and Assembly, Annotation, Analysis, and IT and Informatics. The current versions of the resulting standards documents are available on the EBP website, with the recognition that opportunities, technologies, and challenges may improve or change in the future, requiring flexibility for the EBP to meet its goals. Here, we describe some highlights from the proposed standards, and areas where additional challenges will need to be met.

Ethical, legal, and social issues in the Earth BioGenome Project.

The Earth BioGenome Project (EBP) is an audacious endeavor to obtain whole-genome sequences of representatives from all eukaryotic species on Earth. In addition to the project's technical and organizational challenges, it also faces complicated ethical, legal, and social issues. This paper, from members of the EBP's Ethical, Legal, and Social Issues (ELSI) Committee, catalogs these ELSI concerns arising from EBP. These include legal issues, such as sample collection and permitting; the applicability of international treaties, such as the Convention on Biological Diversity and the Nagoya Protocol; intellectual property; sample accessioning; and biosecurity and ethical issues, such as sampling from the territories of Indigenous peoples and local communities, the protection of endangered species, and cross-border collections, among several others. We also comment on the intersection of digital sequence information and data rights. More broadly, this list of ethical, legal, and social issues for large-scale genomic sequencing projects may be useful in the consideration of ethical frameworks for future projects. While we do not-and cannot-provide simple, overarching solutions for all the issues raised here, we conclude our perspective by beginning to chart a path forward for EBP's work.

Platypus and echidna genomes reveal mammalian biology and evolution.

Egg-laying mammals (monotremes) are the only extant mammalian outgroup to therians (marsupial and eutherian animals) and provide key insights into mammalian evolution1,2. Here we generate and analyse reference genomes of the platypus (Ornithorhynchus anatinus) and echidna (Tachyglossus aculeatus), which represent the only two extant monotreme lineages. The nearly complete platypus genome assembly has anchored almost the entire genome onto chromosomes, markedly improving the genome continuity and gene annotation. Together with our echidna sequence, the genomes of the two species allow us to detect the ancestral and lineage-specific genomic changes that shape both monotreme and mammalian evolution. We provide evidence that the monotreme sex chromosome complex originated from an ancestral chromosome ring configuration. The formation of such a unique chromosome complex may have been facilitated by the unusually extensive interactions between the multi-X and multi-Y chromosomes that are shared by the autosomal homologues in humans. Further comparative genomic analyses unravel marked differences between monotremes and therians in haptoglobin genes, lactation genes and chemosensory receptor genes for smell and taste that underlie the ecological adaptation of monotremes.

Population genomics of the critically endangered kakapo.

The kakapo is a flightless parrot endemic to New Zealand. Once common in the archipelago, only 201 individuals remain today, most of them descending from an isolated island population. We report the first genome-wide analyses of the species, including a high-quality genome assembly for kakapo, one of the first chromosome-level reference genomes sequenced by the Vertebrate Genomes Project (VGP). We also sequenced and analyzed 35 modern genomes from the sole surviving island population and 14 genomes from the extinct mainland population. While theory suggests that such a small population is likely to have accumulated deleterious mutations through genetic drift, our analyses on the impact of the long-term small population size in kakapo indicate that present-day island kakapo have a reduced number of harmful mutations compared to mainland individuals. We hypothesize that this reduced mutational load is due to the island population having been subjected to a combination of genetic drift and purging of deleterious mutations, through increased inbreeding and purifying selection, since its isolation from the mainland ∼10,000 years ago. Our results provide evidence that small populations can survive even when isolated for hundreds of generations. This work provides key insights into kakapo breeding and recovery and more generally into the application of genetic tools in conservation efforts for endangered species.

Vertebrate Chromosome Evolution.

The study of chromosome evolution is undergoing a resurgence of interest owing to advances in DNA sequencing technology that facilitate the production of chromosome-scale whole-genome assemblies de novo. This review focuses on the history, methods, discoveries, and current challenges facing the field, with an emphasis on vertebrate genomes. A detailed examination of the literature on the biology of chromosome rearrangements is presented, specifically the relationship between chromosome rearrangements and phenotypic evolution, adaptation, and speciation. A critical review of the methods for identifying, characterizing, and visualizing chromosome rearrangements and computationally reconstructing ancestral karyotypes is presented. We conclude by looking to the future, identifying the enormous technical and scientific challenges presented by the accumulation of hundreds and eventually thousands of chromosome-scale assemblies.

Broad host range of SARS-CoV-2 predicted by comparative and structural analysis of ACE2 in vertebrates.

The novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of COVID-19. The main receptor of SARS-CoV-2, angiotensin I converting enzyme 2 (ACE2), is now undergoing extensive scrutiny to understand the routes of transmission and sensitivity in different species. Here, we utilized a unique dataset of ACE2 sequences from 410 vertebrate species, including 252 mammals, to study the conservation of ACE2 and its potential to be used as a receptor by SARS-CoV-2. We designed a five-category binding score based on the conservation properties of 25 amino acids important for the binding between ACE2 and the SARS-CoV-2 spike protein. Only mammals fell into the medium to very high categories and only catarrhine primates into the very high category, suggesting that they are at high risk for SARS-CoV-2 infection. We employed a protein structural analysis to qualitatively assess whether amino acid changes at variable residues would be likely to disrupt ACE2/SARS-CoV-2 spike protein binding and found the number of predicted unfavorable changes significantly correlated with the binding score. Extending this analysis to human population data, we found only rare (frequency <0.001) variants in 10/25 binding sites. In addition, we found significant signals of selection and accelerated evolution in the ACE2 coding sequence across all mammals, and specific to the bat lineage. Our results, if confirmed by additional experimental data, may lead to the identification of intermediate host species for SARS-CoV-2, guide the selection of animal models of COVID-19, and assist the conservation of animals both in native habitats and in human care.

Broad Host Range of SARS-CoV-2 Predicted by Comparative and Structural Analysis of ACE2 in Vertebrates.

The novel coronavirus SARS-CoV-2 is the cause of Coronavirus Disease-2019 (COVID-19). The main receptor of SARS-CoV-2, angiotensin I converting enzyme 2 (ACE2), is now undergoing extensive scrutiny to understand the routes of transmission and sensitivity in different species. Here, we utilized a unique dataset of 410 vertebrates, including 252 mammals, to study cross-species conservation of ACE2 and its likelihood to function as a SARS-CoV-2 receptor. We designed a five-category ranking score based on the conservation properties of 25 amino acids important for the binding between receptor and virus, classifying all species from very high to very low. Only mammals fell into the medium to very high categories, and only catarrhine primates in the very high category, suggesting that they are at high risk for SARS-CoV-2 infection. We employed a protein structural analysis to qualitatively assess whether amino acid changes at variable residues would be likely to disrupt ACE2/SARS-CoV-2 binding, and found the number of predicted unfavorable changes significantly correlated with the binding score. Extending this analysis to human population data, we found only rare (<0.1%) variants in 10/25 binding sites. In addition, we observed evidence of positive selection in ACE2 in multiple species, including bats. Utilized appropriately, our results may lead to the identification of intermediate host species for SARS-CoV-2, justify the selection of animal models of COVID-19, and assist the conservation of animals both in native habitats and in human care.

Precision nomenclature for the new genomics.

The confluence of two scientific disciplines may lead to nomenclature conflicts that require new terms while respecting historical definitions. This is the situation with the current state of cytology and genomics, which offer examples of distinct nomenclature and vocabularies that require reconciliation. In this article, we propose the new terms C-scaffold (for chromosome-scale assemblies of sequenced DNA fragments, commonly named scaffolds) and scaffotype (the resulting collection of C-scaffolds that represent an organism's genome). This nomenclature avoids conflict with the historical definitions of the terms chromosome (a microscopic body made of DNA and protein) and karyotype (the collection of images of all chromosomes of an organism or species). As large-scale sequencing projects progress, adoption of this nomenclature will assist end users to properly classify genome assemblies, thus facilitating genomic analysis.