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Studies of Y Chromosome evolution have focused primarily on gene decay, a consequence of suppression of crossing-over with the X Chromosome. Here, we provide evidence that suppression of X–Y crossing-over unleashed a second dynamic: selfish X–Y arms races that reshaped the sex chromosomes in mammals as different as cattle, mice, and men. Using super-resolution sequencing, we explore the Y Chromosome of Bos taurus (bull) and find it to be dominated by massive, lineage-specific amplification of testis-expressed gene families, making it the most gene-dense Y Chromosome sequenced to date. As in mice, an X-linked homolog of a bull Y-amplified gene has become testis-specific and amplified. This evolutionary convergence implies that lineage-specific X–Y coevolution through gene amplification, and the selfish forces underlying this phenomenon, were dominatingly powerful among diverse mammalian lineages. Together with Y gene decay, X–Y arms races molded mammalian sex chromosomes and influenced the course of mammalian evolution.

Most human genetic variation is classified as VUS - variants of uncertain significance. While advances in genome editing have allowed innovation in pooled screening platforms, many screens deal with relatively simple readouts (viability, fluorescence) and cannot identify the complex cellular phenotypes that underlie most human diseases. In this paper, we present a generalizable functional genomics platform that combines high-content imaging, machine learning, and microraft isolation in a new method termed “Raft-Seq”. We highlight the efficacy of our platform by showing its ability to distinguish pathogenic point mutations of the mitochondrial regulator MFN2, even when the cellular phenotype is subtle. We also show that our platform achieves its efficacy using multiple cellular features, which can be configured on-the-fly. Raft-Seq enables a new way to perform pooled screening on sets of mutations in biologically relevant cells, with the ability to physically capture any cell with a perturbed phenotype and expand it clonally, directly from the primary screen.Graphical AbstractHere, we address the need to evaluate the impact of numerous genetic variants. This manuscript depicts the methods of using machine learning on a biologically relevant phenotype to predict specific point mutations, followed by physically capturing those mutated cells.

After birds diverged from mammals, different ancestral autosomes evolved into sex chromosomes in each lineage. In birds, females are ZW and males are ZZ, but in mammals females are XX and males are XY. We sequenced the chicken W chromosome, compared its gene content with our reconstruction of the ancestral autosomes, and followed the evolutionary trajectory of ancestral W-linked genes across birds. Avian W chromosomes evolved in parallel with mammalian Y chromosomes, preserving ancestral genes through selection to maintain the dosage of broadly expressed regulators of key cellular processes. We propose that, like the human Y chromosome, the chicken W chromosome is essential for embryonic viability of the heterogametic sex. Unlike other sequenced sex chromosomes, the chicken W chromosome did not acquire and amplify genes specifically expressed in reproductive tissues. We speculate that the pressures that drive the acquisition of reproduction-related genes on sex chromosomes may be specific to the male germ line.

We sequenced the MSY (male-specific region of the Y chromosome) of the C57BL/6J strain of the laboratory mouse Mus musculus. In contrast to theories that Y chromosomes are heterochromatic and gene poor, the mouse MSY is 99.9% euchromatic and contains about 700 protein-coding genes. Only 2% of the MSY derives from the ancestral autosomes that gave rise to the mammalian sex chromosomes. Instead, all but 45 of the MSY’s genes belong to three acquired, massively amplified gene families that have no homologs on primate MSYs but do have acquired, amplified homologs on the mouse X chromosome. The complete mouse MSY sequence brings to light dramatic forces in sex chromosome evolution: lineage-specific convergent acquisition and amplification of X-Y gene families, possibly fueled by antagonism between acquired X-Y homologs. The mouse MSY sequence presents opportunities for experimental studies of a sex-specific chromosome in its entirety, in a genetically tractable model organism., National Institutes of Health (U.S.), Howard Hughes Medical Institute

The human X and Y chromosomes evolved from an ordinary pair of autosomes, but millions of years ago genetic decay ravaged the Y chromosome, and only three per cent of its ancestral genes survived. We reconstructed the evolution of the Y chromosome across eight mammals to identify biases in gene content and the selective pressures that preserved the surviving ancestral genes. Our findings indicate that survival was nonrandom, and in two cases, convergent across placental and marsupial mammals. We conclude that the gene content of the Y chromosome became specialized through selection to maintain the ancestral dosage of homologous X-Y gene pairs that function as broadly expressed regulators of transcription, translation and protein stability. We propose that beyond its roles in testis determination and spermatogenesis, the Y chromosome is essential for male viability, and has unappreciated roles in Turner's syndrome and in phenotypic differences between the sexes in health and disease.

This evolutionary decay was driven by a series of five ‘stratification’ events. Each event suppressed X–Y crossing over within a chromosome segment or ‘stratum’, incorporated that segment into the MSY and subjected its genes to the erosive forces that attend the absence of crossing over 2,6 . The last of these events occurred 30 million years ago, 5 million years before the human and Old World monkey lineages diverged. Although speculation abounds regarding ongoing decay and looming extinction of the human Y chromosome 7–10 , remarkably little is known about how many MSY genes were lost in the human lineage in the 25 million years that have followed its separation from the Old World monkey lineage. To investigate this question, we sequenced the MSY of the rhesus macaque, an Old World monkey, and compared it to the human MSY. We discovered that during the last 25 million years MSY gene loss in the human lineage was limited to the youngest stratum (stratum 5), which comprises three percent of the human MSY. In the older strata, which collectively comprise the bulk of the human MSY, gene loss evidently ceased more than 25 million years ago. Likewise, the rhesus MSY has not lost any older genes (from strata 1–4) during the past 25 million years, despite its major structural differences to the human MSY. The rhesus MSY is simpler, with few amplified gene families or palindromes that might enable intrachromosomal recombination and repair. We present an empirical reconstruction of human MSY evolution in which each stratum transitioned from rapid, exponential loss of ancestral genes to strict conservation through purifying selection. The human Y chromosome no longer engages in crossing over with its once-identical partner, the X chromosome, except in its pseudoautosomal regions. During evolution, X–Y crossing over was suppressed in five different chromosomal regions at five different times, each probably resulting from an inversion in the Y chromosome 2,3 . Each of these regions of the Y chromosome then began its own individual course of degeneration, experiencing deletions and gene loss. Comparison of the present-day X and Y chromosomes enables identification of these five evolutionary ‘strata’ in the MSY (and X chromosome); their distinctive degrees of X–Y differentiation indicate their evolutionary ages 2,3 . The oldest stratum (stratum 1) dates back over 240 million years (Myr) 2 and is the most highly differentiated, and the youngest stratum (stratum 5) originated only 30 Myr ago and displays the highest X–Y nucleotide sequence similarity within the MSY 3 . The five strata and their respective decay processes, over tens to hundreds of millions of years of mammalian evolution, offer replicate experiments of nature from which to reconstruct the trajectories and kinetics of gene loss in the MSY. Only the human and chimpanzee MSYs had been sequenced before the present study, and they are separated by just 6 Myr of evolution. We decided to examine the MSY of a much more distant relative, the rhesus macaque (Macaca mulatta), to enable us to reconstruct gene loss and conservation in the MSY during the past 25 Myr. We sequenced the rhesus MSY using bacterial artificial chromosome (BAC) clones and the SHIMS (single-haplotype iterative mapping and sequencing) strategy that has previously been used in the human and chimpanzee MSYs 4,11–13 as well as in the chicken Z chromosome 5 . The resulting sequence is comprised of 11.0 megabases (Mb), is complete aside from three small gaps and has an error rate of about one nucleotide per Mb. We ordered and oriented the finished sequence contigs by fluorescence in situ hybridization and radiation hybrid mapping (Supplementary Figs 1–6, Supplementary Table 1, Supplemen

When the platypus (Ornithorhynchus anatinus) was first discovered, it was thought to be a taxidermist’s hoax, as it has a blend of mammalian and reptilian features. It is a most remarkable mammal, not only because it lays eggs but also because it is venomous. Rather than delivering venom through a bite, as do snakes and shrews, male platypuses have venomous spurs on each hind leg. The platypus genome sequence provides a unique opportunity to unravel the evolutionary history of many of these interesting features. While searching the platypus genome for the sequences of antimicrobial defensin genes, we identified three Ornithorhynchus venom defensin-like peptide (OvDLP) genes, which produce the major components of platypus venom. We show that gene duplication and subsequent functional diversification of beta-defensins gave rise to these platypus OvDLPs. The OvDLP genes are located adjacent to the beta-defensins and share similar gene organization and peptide structures. Intriguingly, some species of snakes and lizards also produce venoms containing similar molecules called crotamines and crotamine-like peptides. This led us to trace the evolutionary origins of other components of platypus and reptile venom. Here we show that several venom components have evolved separately in the platypus and reptiles. Convergent evolution has repeatedly selected genes coding for proteins containing specific structural motifs as templates for venom molecules.

In mammals, with a few notable exceptions, sex is determined by an XX female:XY male sex chromosome system in which the Y chromosome bears the male-dominant testis-determining gene SRY. The X chromosome of most placental mammals has a virtually identical gene content, whereas the degenerate Y chromosome contains overlapping subsets of only a few active genes (for review, see Graves 2006). Marsupial mammals have a smaller X and Y that represent an ancestral therian mammal sex pair and define the ancient region of the human sex chromosomes. The mammal X and Y evolved from a pair of autosomes after the proto-Y acquired a male-determining gene, X–Y recombination between male-advantage genes was suppressed, and the Y degenerated (Charlesworth et al. 2005; Fraser and Heitman 2005; Graves 2006). Mapping orthologs of mammal X-borne genes in other vertebrates will help our understanding of how the mammal XY and the sex-determining (SRY) gene evolved, and also how they function. In other amniotes (reptiles and birds), sex is determined by a variety of different mechanisms that belong in two broad classes: genetic or environmental, although recent data show that some species combine both (Quinn et al. 2007). Genetic sex determination is the most widespread within lineages and commonly involves a pair of differentiated sex chromosomes (for review, see Ezaz et al. 2006). Sex chromosomes are named XY when the heterogametic sex is the male (female XX and male XY as in mammals) and ZW when heterogametic sex is the female (female ZW and male ZZ as in birds or snakes). Amniote XY and ZW sex chromosome pairs are superficially similar. The paired chromosome (X or Z) in the homogametic sex is generally well conserved, large, and gene-rich, whereas the sex-specific Y or W is usually small, heterochromatic, and almost devoid of active genes. However, comparative gene mapping shows that the sex chromosomes of mammals (XY), birds (ZW), and snakes (ZW) are entirely nonhomologous, implying that they evolved from different pairs of autosomes (Fridolfsson et al. 1998; Nanda et al. 2000; Graves and Shetty 2001; Kohn et al. 2004; Matsubara et al. 2006). The sex-determining gene that initiates testis development is also different; for instance, there is no SRY in non-mammal vertebrates, and sex determination in birds seems to be largely controlled by dosage of a Z-borne gene (possibly DMRT1) (Raymond et al. 1999) rather than a male-dominant gene. The similarities shared by sex chromosomes are thus the results of convergent evolutionary forces in different vertebrate lineages. The acquisition of a sex-determining gene on one member of an autosome pair and the accumulation of sex-specific alleles nearby were followed by suppression of recombination between the new proto-sex chromosomes, which resulted in vulnerability to mutation and deletions of the sex-specific chromosome (Y or W) and, therefore, its degradation (Charlesworth and Charlesworth 2000; Charlesworth et al. 2005; Fraser and Heitman 2005; Steinemann and Steinemann 2005; Graves 2006). The original autosome from which the mammal X and Y diverged can be deduced from the relationship of the X pair to other vertebrate sex chromosome systems. The short arm of chromosome 4 in the chicken (Gallus gallus [GGA]) and several autosomal regions in fish share genes with the mammal X, and thus represent this ancestral autosome. How this genomic region took over a sex-determining function from an ancestral temperature-determining system, or from nonhomologous ancestral sex chromosomes, demands exploration of the sex-determining system of the most basal mammal group, the monotremes. Monotremes diverged ∼165 million years ago (Mya) from therian mammals (placentals and marsupials) and therefore fall in the phylogenetic tree between birds/reptiles and therians (Van Rheede et al. 2006; Bininda-Emonds et al. 2007). Monotremes, represented only by the duck-billed platypus Ornithorhynchus anatinus and the echidnas (four species), display a fascinating mixture of mammalian and avian/reptilian morphological, physiological, and karyological features and have a unique reproductive system that combines egg-laying with lactation (for review, see Grutzner and Graves 2004). Monotremes have long been known to possess a complex male heterogametic system in which multiple X and Y chromosomes form a chain at male meiosis (Bick and Sharman 1975; Murtagh 1977; Wrigley and Graves 1988). Recently, individual X and Y chromosomes were identified by chromosome painting (Rens et al. 2004). The male was discovered to have 10 unpaired chromosomes that included five male-specific Y chromosomes and five X chromosomes; the female possesses two copies of the five Xs. In male meiosis, the 10 sex chromosomes form an alternating XY chain, X1Y1X2Y2X3Y3X4Y4X5Y5 (Grutzner et al. 2004), unique in vertebrates (for review, see Grutzner et al. 2006). These 10 chromosomes pair and recombine in pseudoautosomal regions at the termini of adjacent X and Y chromosomes. Little is known about the gene content of the 10 platypus sex chromosomes, but the few available data are extremely striking. Early comparative mapping using radioactive in situ hybridization with heterologous probes suggested that X1, located at one end of the chain, shared homology with the ancient part of the mammalian X (Watson et al. 1990; Wilcox et al. 1996; Mitchell et al. 1998; but see also Waters et al. 2005). At the other end of the chain, fluorescent in situ hybridization (FISH) of a large insert BAC-clone showed that X5 contained the Z-borne putative bird sex-determination gene DMRT1 (Grutzner et al. 2004; El-Mogharbel et al. 2007). This suggested that the monotreme meiotic chain has homology with the therian XY system at one end and to the bird ZW system at the other, and represents an evolutionary link between two systems that were previously thought to have evolved independently (Grutzner et al. 2004; Ezaz et al. 2006). The identity of the platypus sex-determining gene remains mysterious. Many efforts to identify a platypus homolog of the therian testis-determining gene SRY proved fruitless (Grutzner et al. 2004). Progress in platypus genome sequencing and the availability of BAC clones whose gene content can be identified from the database now allow us to partially characterize the gene content of X specific regions and identify genes on the pairing regions between adjacent X and Y chromosomes. We have also tested the hypotheses that platypus sex chromosomes share homology with both the mammal XY and the bird ZW systems. In complete contradiction to early data, we find that the 10 sex chromosomes of platypus share no homology with the ancestral therian X chromosome, which is homologous to platypus chromosome 6. Instead, we find that regions homologous to the chicken Z are scattered throughout the chain, principally on X5 and X3. These results have major implications for our understanding of mammalian sex chromosome evolution.

I have read the journal's policy and have the following conflicts: Paul Flicek is married to the deputy editor of PLoS Medicine, Melissa Norton. Evan Eichler is on the board of Pacific Biosciences. Support for this work came from the Intramural Research Program of the NIH, The National Library of Medicine, the European Molecular Biology Laboratory, the Wellcome Trust (grant number 077198), and the Howard Hughes Medical Institute (EEE). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

In birds, as in mammals, one pair of chromosomes differs between the sexes. In birds, males are ZZ and females ZW. In mammals, males are XY and females XX. Like the mammalian XY pair, the avian ZW pair is believed to have evolved from autosomes, with most change occurring in the chromosomes found in only one sex—the W and Y chromosomes1, 2, 3, 4, 5. By contrast, the sex chromosomes found in both sexes—the Z and X chromosomes—are assumed to have diverged little from their autosomal progenitors2. Here we report findings that challenge this assumption for both the chicken Z chromosome and the human X chromosome. The chicken Z chromosome, which we sequenced essentially to completion, is less gene-dense than chicken autosomes but contains a massive tandem array containing hundreds of duplicated genes expressed in testes. A comprehensive comparison of the chicken Z chromosome with the finished sequence of the human X chromosome demonstrates that each evolved independently from different portions of the ancestral genome. Despite this independence, the chicken Z and human X chromosomes share features that distinguish them from autosomes: the acquisition and amplification of testis-expressed genes, and a low gene density resulting from an expansion of intergenic regions. These features were not present on the autosomes from which the Z and X chromosomes originated but were instead acquired during the evolution of Z and X as sex chromosomes. We conclude that the avian Z and mammalian X chromosomes followed convergent evolutionary trajectories, despite their evolving with opposite (female versus male) systems of heterogamety. More broadly, in birds and mammals, sex chromosome evolution involved not only gene loss in sex-specific chromosomes, but also marked expansion and gene acquisition in sex chromosomes common to males and females., National Science Foundation (U.S.), Howard Hughes Medical Institute

International audience; We present a draft genome sequence of the platypus, Ornithorhynchus anatinus. This monotreme exhibits a fascinating combination of reptilian and mammalian characters. For example, platypuses have a coat of fur adapted to an aquatic lifestyle; platypus females lactate, yet lay eggs; and males are equipped with venom similar to that of reptiles. Analysis of the first monotreme genome aligned these features with genetic innovations. We find that reptile and platypus venom proteins have been co- opted independently from the same gene families; milk protein genes are conserved despite platypuses laying eggs; and immune gene family expansions are directly related to platypus biology. Expansions of protein, non- protein- coding RNA and microRNA families, as well as repeat elements, are identified. Sequencing of this genome now provides a valuable resource for deep mammalian comparative analyses, as well as for monotreme biology and conservation.

In mammals, chromosomes occupy defined positions in sperm, whereas previous work in chicken showed random chromosome distribution. Monotremes (platypus and echidnas) are the most basal group of living mammals. They have elongated sperm like chicken and a complex sex chromosome system with homology to chicken sex chromosomes. We used platypus and chicken genomic clones to investigate genome organization in sperm. In chicken sperm, about half of the chromosomes investigated are organized non-randomly, whereas in platypus chromosome organization in sperm is almost entirely non-random. The use of genomic clones allowed us to determine chromosome orientation and chromatin compaction in sperm. We found that in both species chromosomes maintain orientation of chromosomes in sperm independent of random or non-random positioning along the sperm nucleus. The distance of loci correlated with the total length of sperm nuclei, suggesting that chromatin extension depends on sperm elongation. In platypus, most sex chromosomes cluster in the posterior region of the sperm nucleus, presumably the result of postmeiotic association of sex chromosomes. Chicken and platypus autosomes sharing homology with the human X chromosome located centrally in both species suggesting that this is the ancestral position. This suggests that in some therian mammals a more anterior position of the X chromosome has evolved independently.

Like the unique platypus itself, the platypus genome is extraordinary because of its complex sex chromosome system, and is controversial because of difficulties in identification of small autosomes and sex chromosomes. A 6-fold shotgun sequence of the platypus genome is now available and is being assembled with the help of physical mapping. It is therefore essential to characterize the chromosomes and resolve the ambiguities and inconsistencies in identifying autosomes and sex chromosomes. We have used chromosome paints and DAPI banding to identify and classify pairs of autosomes and sex chromosomes. We have established an agreed nomenclature and identified anchor BAC clones for each chromosome that will ensure unambiguous gene localizations.

Human chromosome 2 is unique to the human lineage in being the product of a head-to-head fusion of two intermediate-sized ancestral chromosomes. Chromosome 4 has received attention primarily related to the search for the Huntington's disease gene, but also for genes associated with Wolf-Hirschhorn syndrome, polycystic kidney disease and a form of muscular dystrophy. Here we present approximately 237 million base pairs of sequence for chromosome 2, and 186 million base pairs for chromosome 4, representing more than 99.6% of their euchromatic sequences. Our initial analyses have identified 1,346 protein-coding genes and 1,239 pseudogenes on chromosome 2, and 796 protein-coding genes and 778 pseudogenes on chromosome 4. Extensive analyses confirm the underlying construction of the sequence, and expand our understanding of the structure and evolution of mammalian chromosomes, including gene deserts, segmental duplications and highly variant regions.

Strategies for assembling large, complex genomes have evolved to include a combination of whole-genome shotgun sequencing and hierarchal map-assisted sequencing1, 2. Whole-genome maps of all types can aid genome assemblies, generally starting with low-resolution cytogenetic maps and ending with the highest resolution of sequence. Fingerprint clone maps are based upon complete restriction enzyme digests of clones representative of the target genome, and ultimately comprise a near-contiguous path of clones across the genome. Such clone-based maps are used to validate sequence assembly order, supply long-range linking information for assembled sequences, anchor sequences to the genetic map and provide templates for closing gaps. Fingerprint maps are also a critical resource for subsequent functional genomic studies, because they provide a redundant and ordered sampling of the genome with clones3. In an accompanying paper4 we describe the draft genome sequence of the chicken, Gallus gallus, the first species sequenced that is both a model organism and a global food source. Here we present a clone-based physical map of the chicken genome at 20-fold coverage, containing 260 contigs of overlapping clones. This map represents approximately 91% of the chicken genome and enables identification of chicken clones aligned to positions in other sequenced genomes

A physical map of a genome is an essential guide for navigation, allowing the location of any gene or other landmark in the chromosomal DNA. We have constructed a physical map of the mouse genome that contains 296 contigs of overlapping bacterial clones and 16,992 unique markers. The mouse contigs were aligned to the human genome sequence on the basis of 51,486 homology matches, thus enabling use of the conserved synteny (correspondence between chromosome blocks) of the two genomes to accelerate construction of the mouse map. The map provides a framework for assembly of whole-genome shotgun sequence data, and a tile path of clones for generation of the reference sequence. Definition of the human-mouse alignment at this level of resolution enables identification of a mouse clone that corresponds to almost any position in the human genome. The human sequence may be used to facilitate construction of other mammalian genome maps using the same strategy.

Nature 508, 494–499 (2014); doi:10.1038/nature13206 Jessica Alfoldi should have been listed with affiliation 1 in the author list. She performed BAC mapping, radiation hybrid mapping and real-time polymerase chain reaction analyses. The online versions of this Article have been corrected.

Nature 453, 175–183 (2008) In this Article, Mikhail Nefedov and Pieter J. de Jong were omitted from the author list.