Evolution and expression patterns of the neo-sex chromosomes of the crested ibis.

Bird sex chromosomes play a unique role in sex-determination, and affect the sexual morphology and behavior of bird species. Core waterbirds, a major clade of birds, share the common characteristics of being sexually monomorphic and having lower levels of inter-sexual conflict, yet their sex chromosome evolution remains poorly understood. Here, by we analyse of a chromosome-level assembly of a female crested ibis (Nipponia nippon), a typical core waterbird. We identify neo-sex chromosomes resulting from fusion of microchromosomes with ancient sex chromosomes. These fusion events likely occurred following the divergence of Threskiornithidae and Ardeidae. The neo-W chromosome of the crested ibis exhibits the characteristics of slow degradation, which is reflected in its retention of abundant gametologous genes. Neo-W chromosome genes display an apparent ovary-biased gene expression, which is largely driven by genes that are retained on the crested ibis W chromosome but lost in other bird species. These results provide new insights into the evolutionary history and expression patterns for the sex chromosomes of bird species.

A pangenome graph reference of 30 chicken genomes allows genotyping of large and complex structural variants.

BACKGROUND: The red junglefowl, the wild outgroup of domestic chickens, has historically served as a reference for genomic studies of domestic chickens. These studies have provided insight into the etiology of traits of commercial importance. However, the use of a single reference genome does not capture diversity present among modern breeds, many of which have accumulated molecular changes due to drift and selection. While reference-based resequencing is well-suited to cataloging simple variants such as single-nucleotide changes and short insertions and deletions, it is mostly inadequate to discover more complex structural variation in the genome. METHODS: We present a pangenome for the domestic chicken consisting of thirty assemblies of chickens from different breeds and research lines. RESULTS: We demonstrate how this pangenome can be used to catalog structural variants present in modern breeds and untangle complex nested variation. We show that alignment of short reads from 100 diverse wild and domestic chickens to this pangenome reduces reference bias by 38%, which affects downstream genotyping results. This approach also allows for the accurate genotyping of a large and complex pair of structural variants at the K feathering locus using short reads, which would not be possible using a linear reference. CONCLUSIONS: We expect that this new paradigm of genomic reference will allow better pinpointing of exact mutations responsible for specific phenotypes, which will in turn be necessary for breeding chickens that meet new sustainability criteria and are resilient to quickly evolving pathogen threats.

A high-quality genome assembly highlights the evolutionary history of the great bustard (Otis tarda, Otidiformes).

Conservation genomics often relies on non-invasive methods to obtain DNA fragments which limit the power of multi-omic analyses for threatened species. Here, we report multi-omic analyses based on a well-preserved great bustard individual (Otis tarda, Otidiformes) that was found dead in the mountainous region in Gansu, China. We generate a near-complete genome assembly containing only 18 gaps scattering in 8 out of the 40 assembled chromosomes. We characterize the DNA methylation landscape which is correlated with GC content and gene expression. Our phylogenomic analysis suggests Otidiformes and Musophagiformes are sister groups that diverged from each other 46.3 million years ago. The genetic diversity of great bustard is found the lowest among the four available Otidiformes genomes, possibly due to population declines during past glacial periods. As one of the heaviest migratory birds, great bustard possesses several expanded gene families related to cardiac contraction, actin contraction, calcium ion signaling transduction, as well as positively selected genes enriched for metabolism. Finally, we identify an extremely young evolutionary stratum on the sex chromosome, a rare case among birds. Together, our study provides insights into the conservation genomics, adaption and chromosome evolution of the great bustard.

Three amphioxus reference genomes reveal gene and chromosome evolution of chordates.

The slow-evolving invertebrate amphioxus has an irreplaceable role in advancing our understanding of the vertebrate origin and innovations. Here we resolve the nearly complete chromosomal genomes of three amphioxus species, one of which best recapitulates the 17 chordate ancestor linkage groups. We reconstruct the fusions, retention, or rearrangements between descendants of whole-genome duplications, which gave rise to the extant microchromosomes likely existed in the vertebrate ancestor. Similar to vertebrates, the amphioxus genome gradually establishes its three-dimensional chromatin architecture at the onset of zygotic activation and forms two topologically associated domains at the Hox gene cluster. We find that all three amphioxus species have ZW sex chromosomes with little sequence differentiation, and their putative sex-determining regions are nonhomologous to each other. Our results illuminate the unappreciated interspecific diversity and developmental dynamics of amphioxus genomes and provide high-quality references for understanding the mechanisms of chordate functional genome evolution.

Evolutionary analysis of a complete chicken genome.

Microchromosomes are prevalent in nonmammalian vertebrates [P. D. Waters et al., Proc. Natl. Acad. Sci. U.S.A. 118 (2021)], but a few of them are missing in bird genome assemblies. Here, we present a new chicken reference genome containing all autosomes, a Z and a W chromosome, with all gaps closed except for the W. We identified ten small microchromosomes (termed dot chromosomes) with distinct sequence and epigenetic features, among which six were newly assembled. Those dot chromosomes exhibit extremely high GC content and a high level of DNA methylation and are enriched for housekeeping genes. The pericentromeric heterochromatin of dot chromosomes is disproportionately large and continues to expand with the proliferation of satellite DNA and testis-expressed genes. Our analyses revealed that the 41-bp CNM repeat frequently forms higher-order repeats (HORs) at the centromeres of acrocentric chromosomes. The centromere core regions where the kinetochore attaches often encompass telomeric sequence (TTAGGG)n, and in a one of the dot chromosomes, the centromere core recruits an endogenous retrovirus (ERV). We further demonstrate that the W chromosome shares some common features with dot chromosomes, having large arrays of hypermethylated tandem repeats. Finally, using the complete chicken chromosome models, we reconstructed a fine picture of chordate karyotype evolution, revealing frequent chromosomal fusions before and after vertebrate whole-genome duplications. Our sequence and epigenetic characterization of chicken chromosomes shed insights into the understanding of vertebrate genome evolution and chromosome biology.

Independent Evolution of Sex Chromosomes and Male Pregnancy-Related Genes in Two Seahorse Species.

Unlike birds and mammals, many teleosts have homomorphic sex chromosomes, and changes in the chromosome carrying the sex-determining locus, termed "turnovers", are common. Recent turnovers allow studies of several interesting questions. One question is whether the new sex-determining regions evolve to become completely non-recombining, and if so, how and why. Another is whether (as predicted) evolutionary changes that benefit one sex accumulate in the newly sex-linked region. To study these questions, we analyzed the genome sequences of two seahorse species of the Syngnathidae, a fish group in which many species evolved a unique structure, the male brood pouch. We find that both seahorse species have XY sex chromosome systems, but their sex chromosome pairs are not homologs, implying that at least one turnover event has occurred. The Y-linked regions occupy 63.9% and 95.1% of the entire sex chromosome of the two species and do not exhibit extensive sequence divergence with their X-linked homologs. We find evidence for occasional recombination between the extant sex chromosomes that may account for their homomorphism. We argue that these Y-linked regions did not evolve by recombination suppression after the turnover, but by the ancestral nature of the low crossover rates in these chromosome regions. With such an ancestral crossover landscape, a turnover can instantly create an extensive Y-linked region. Finally, we test for adaptive evolution of male pouch-related genes after they became Y-linked in the seahorse.

Joint profiling of gene expression and chromatin accessibility during amphioxus development at single-cell resolution.

Vertebrate evolution was accompanied by two rounds of whole-genome duplication followed by functional divergence in terms of regulatory circuits and gene expression patterns. As a basal and slow-evolving chordate species, amphioxus is an ideal paradigm for exploring the origin and evolution of vertebrates. Single-cell sequencing has been widely used to construct the developmental cell atlas of several representative species of vertebrates (human, mouse, zebrafish, and frog) and tunicates (sea squirts). Here, we perform single-nucleus RNA sequencing (snRNA-seq) and single-cell assay for transposase accessible chromatin sequencing (scATAC-seq) for different stages of amphioxus (covering embryogenesis and adult tissues). With the datasets generated, we constructed a developmental tree for amphioxus cell fate commitment and lineage specification and characterize the underlying key regulators and genetic regulatory networks. The data are publicly available on the online platform AmphioxusAtlas.

Recurrent chromosome reshuffling and the evolution of neo-sex chromosomes in parrots.

The karyotype of most birds has remained considerably stable during more than 100 million years' evolution, except for some groups, such as parrots. The evolutionary processes and underlying genetic mechanism of chromosomal rearrangements in parrots, however, are poorly understood. Here, using chromosome-level assemblies of four parrot genomes, we uncover frequent chromosome fusions and fissions, with most of them occurring independently among lineages. The increased activities of chromosomal rearrangements in parrots are likely associated with parrot-specific loss of two genes, ALC1 and PARP3, that have known functions in the repair of double-strand breaks and maintenance of genome stability. We further find that the fusion of the ZW sex chromosomes and chromosome 11 has created a pair of neo-sex chromosomes in the ancestor of parrots, and the chromosome 25 has been further added to the sex chromosomes in monk parakeet. Together, the combination of our genomic and cytogenetic analyses characterizes the complex evolutionary history of chromosomal rearrangements and sex chromosomes in parrots.

Phylogeny and sex chromosome evolution of Palaeognathae.

Many paleognaths (ratites and tinamous) have a pair of homomorphic ZW sex chromosomes in contrast to the highly differentiated sex chromosomes of most other birds. To understand the evolutionary causes for the different tempos of sex chromosome evolution, we produced female genomes of 12 paleognathous species and reconstructed the phylogeny and the evolutionary history of paleognathous sex chromosomes. We uncovered that Palaeognathae sex chromosomes had undergone stepwise recombination suppression and formed a pattern of "evolutionary strata". Nine of the 15 studied species' sex chromosomes have maintained homologous recombination in their long pseudoautosomal regions extending more than half of the entire chromosome length. We found that in the older strata, the W chromosome suffered more serious functional gene loss. Their homologous Z-linked regions, compared with other genomic regions, have produced an excess of species-specific autosomal duplicated genes that evolved female-specific expression, in contrast to their broadly expressed progenitors. We speculate such "defeminization" of Z chromosome with underrepresentation of female-biased genes and slow divergence of sex chromosomes of paleognaths might be related to their distinctive mode of sexual selection targeting females rather than males, which evolved in their common ancestors.

Telomere-to-telomere assembly of a fish Y chromosome reveals the origin of a young sex chromosome pair.

BACKGROUND: The origin of sex chromosomes requires the establishment of recombination suppression between the proto-sex chromosomes. In many fish species, the sex chromosome pair is homomorphic with a recent origin, providing species for studying how and why recombination suppression evolved in the initial stages of sex chromosome differentiation, but this requires accurate sequence assembly of the X and Y (or Z and W) chromosomes, which may be difficult if they are recently diverged. RESULTS: Here we produce a haplotype-resolved genome assembly of zig-zag eel (Mastacembelus armatus), an aquaculture fish, at the chromosomal scale. The diploid assembly is nearly gap-free, and in most chromosomes, we resolve the centromeric and subtelomeric heterochromatic sequences. In particular, the Y chromosome, including its highly repetitive short arm, has zero gaps. Using resequencing data, we identify a ~7 Mb fully sex-linked region (SLR), spanning the sex chromosome centromere and almost entirely embedded in the pericentromeric heterochromatin. The SLRs on the X and Y chromosomes are almost identical in sequence and gene content, but both are repetitive and heterochromatic, consistent with zero or low recombination. We further identify an HMG-domain containing gene HMGN6 in the SLR as a candidate sex-determining gene that is expressed at the onset of testis development. CONCLUSIONS: Our study supports the idea that preexisting regions of low recombination, such as pericentromeric regions, can give rise to SLR in the absence of structural variations between the proto-sex chromosomes.

A new duck genome reveals conserved and convergently evolved chromosome architectures of birds and mammals.

BACKGROUND: Ducks have a typical avian karyotype that consists of macro- and microchromosomes, but a pair of much less differentiated ZW sex chromosomes compared to chickens. To elucidate the evolution of chromosome architectures between ducks and chickens, and between birds and mammals, we produced a nearly complete chromosomal assembly of a female Pekin duck by combining long-read sequencing and multiplatform scaffolding techniques. RESULTS: A major improvement of genome assembly and annotation quality resulted from the successful resolution of lineage-specific propagated repeats that fragmented the previous Illumina-based assembly. We found that the duck topologically associated domains (TAD) are demarcated by putative binding sites of the insulator protein CTCF, housekeeping genes, or transitions of active/inactive chromatin compartments, indicating conserved mechanisms of spatial chromosome folding with mammals. There are extensive overlaps of TAD boundaries between duck and chicken, and also between the TAD boundaries and chromosome inversion breakpoints. This suggests strong natural selection pressure on maintaining regulatory domain integrity, or vulnerability of TAD boundaries to DNA double-strand breaks. The duck W chromosome retains 2.5-fold more genes relative to chicken. Similar to the independently evolved human Y chromosome, the duck W evolved massive dispersed palindromic structures, and a pattern of sequence divergence with the Z chromosome that reflects stepwise suppression of homologous recombination. CONCLUSIONS: Our results provide novel insights into the conserved and convergently evolved chromosome features of birds and mammals, and also importantly add to the genomic resources for poultry studies.

A new emu genome illuminates the evolution of genome configuration and nuclear architecture of avian chromosomes.

Emu and other ratites are more informative than any other birds in reconstructing the evolution of the ancestral avian or vertebrate karyotype because of their much slower rate of genome evolution. Here, we generated a new chromosome-level genome assembly of a female emu, and estimated the tempo of chromosome evolution across major avian phylogenetic branches, by comparing it to chromosome-level genome assemblies of 11 other bird and one turtle species. We found ratites exhibited the lowest numbers of intra- and inter-chromosomal changes among birds since their divergence with turtles. The small-sized and gene-rich emu microchromosomes have frequent inter-chromosomal contacts that are associated with housekeeping genes, which appears to be driven by clustering their centromeres in the nuclear interior, away from the macrochromosomes in the nuclear periphery. Unlike nonratite birds, only less than one-third of the emu W Chromosome regions have lost homologous recombination and diverged between the sexes. The emu W is demarcated into a highly heterochromatic region (WS0) and another recently evolved region (WS1) with only moderate sequence divergence with the Z Chromosome. WS1 has expanded its inactive chromatin compartment, increased chromatin contacts within the region, and decreased contacts with the nearby regions, possibly influenced by the spreading of heterochromatin from WS0. These patterns suggest that alteration of chromatin conformation comprises an important early step of sex chromosome evolution. Overall, our results provide novel insights into the evolution of avian genome structure and sex chromosomes in three-dimensional space.

High-quality chromosome-level genomes of two tilapia species reveal their evolution of repeat sequences and sex chromosomes.

Tilapias are one of the most farmed fishes that are coined as "aquatic chicken" by the food industry. Nile tilapia and blue tilapia exhibit very recent transition of sex chromosome systems since their divergence approximately five million years ago, making them a great model for elucidating the molecular and evolutionary mechanisms of sex chromosome turnovers. Studies of their sex-determining pathways are also critical for developing genetic sex control in aquaculture. We report here the newly produced genomes of Nile tilapia and blue tilapia that integrate long-read sequencing and chromatin conformation data. The two nearly complete genomes have anchored over 97% of the sequences into linkage groups (LGs), and assembled majorities of complex repetitive regions including telomeres, centromeres and rDNA clusters. In particular, we inferred two episodes of repeat expansion at LG3 respectively in the ancestor of cichlids and that of tilapias. The consequential large heterochromatic region concentrated at one end of LG3 comprises tandem arrays of mRNA and small RNA genes, among which we have identified a candidate female determining gene Paics in blue tilapia. Paics shows female-specific patterns of single-nucleotide variants, copy numbers and expression patterns in gonads during early gonadogenesis. Our work provides a very important genomic resource for functional studies of cichlids, and suggested that unequal distribution of repeat content that impacts the local recombination rate might make some chromosomes more likely to become sex chromosomes.

Identifying the causes and consequences of assembly gaps using a multiplatform genome assembly of a bird-of-paradise.

Genome assemblies are currently being produced at an impressive rate by consortia and individual laboratories. The low costs and increasing efficiency of sequencing technologies now enable assembling genomes at unprecedented quality and contiguity. However, the difficulty in assembling repeat-rich and GC-rich regions (genomic "dark matter") limits insights into the evolution of genome structure and regulatory networks. Here, we compare the efficiency of currently available sequencing technologies (short/linked/long reads and proximity ligation maps) and combinations thereof in assembling genomic dark matter. By adopting different de novo assembly strategies, we compare individual draft assemblies to a curated multiplatform reference assembly and identify the genomic features that cause gaps within each assembly. We show that a multiplatform assembly implementing long-read, linked-read and proximity sequencing technologies performs best at recovering transposable elements, multicopy MHC genes, GC-rich microchromosomes and the repeat-rich W chromosome. Telomere-to-telomere assemblies are not a reality yet for most organisms, but by leveraging technology choice it is now possible to minimize genome assembly gaps for downstream analysis. We provide a roadmap to tailor sequencing projects for optimized completeness of both the coding and noncoding parts of nonmodel genomes.

Origin of a Giant Sex Chromosome.

Chromosome size and morphology vary within and among species, but little is known about the proximate or ultimate causes of these differences. Cichlid fish species in the tribe Oreochromini share an unusual giant chromosome that is ∼3 times longer than the other chromosomes. This giant chromosome functions as a sex chromosome in some of these species. We test two hypotheses of how this giant sex chromosome may have evolved. The first hypothesis proposes that it evolved by accumulating repetitive elements as recombination was reduced around a dominant sex determination locus, as suggested by canonical models of sex chromosome evolution. An alternative hypothesis is that the giant sex chromosome originated via the fusion of an autosome with a highly repetitive B chromosome, one of which carried a sex determination locus. We test these hypotheses using comparative analysis of chromosome-scale cichlid and teleost genomes. We find that the giant sex chromosome consists of three distinct regions based on patterns of recombination, gene and transposable element content, and synteny to the ancestral autosome. The WZ sex determination locus encompasses the last ∼105 Mb of the 134-Mb giant chromosome. The last 47 Mb of the giant chromosome shares no obvious homology to any ancestral chromosome. Comparisons across 69 teleost genomes reveal that the giant sex chromosome contains unparalleled amounts of endogenous retroviral elements, immunoglobulin genes, and long noncoding RNAs. The results favor the B chromosome fusion hypothesis for the origin of the giant chromosome.

Sequence Transpositions Restore Genes on the Highly Degenerated W Chromosomes of Songbirds.

The female-specific W chromosomes of most Neognathae birds are highly degenerated and gene-poor. Previous studies have demonstrated that the gene repertoires of the Neognathae bird W chromosomes, despite being in small numbers, are conserved across bird species, likely due to purifying selection maintaining the regulatory and dosage-sensitive genes. Here we report the discovery of DNA-based sequence duplications from the Z to the W chromosome in birds-of-paradise (Paradisaeidae, Passeriformes), through sequence transposition. The original transposition involved nine genes, but only two of them (ANXA1 and ALDH1A1) survived on the W chromosomes. Both ANXA1 and ALDH1A1 are predicted to be dosage-sensitive, and the expression of ANXA1 is restricted to ovaries in all the investigated birds. These analyses suggest the newly transposed gene onto the W chromosomes can be favored for their role in restoring dosage imbalance or through female-specific selection. After examining seven additional songbird genomes, we further identified five other transposed genes on the W chromosomes of Darwin's finches and one in the great tit, expanding the observation of the Z-to-W transpositions to a larger range of bird species, but not all transposed genes exhibit dosage-sensitivity or ovary-biased expression We demonstrate a new mechanism by which the highly degenerated W chromosomes of songbirds can acquire genes from the homologous Z chromosomes, but further functional investigations are needed to validate the evolutionary forces underlying the transpositions.

The Female-Specific W Chromosomes of Birds Have Conserved Gene Contents but Are Not Feminized.

Sex chromosomes are unique genomic regions with sex-specific or sex-biased inherent patterns and are expected to be more frequently subject to sex-specific selection. Substantial knowledge on the evolutionary patterns of sex-linked genes have been gained from the studies on the male heterogametic systems (XY male, XX female), but the understanding of the role of sex-specific selection in the evolution of female-heterogametic sex chromosomes (ZW female, ZZ male) is limited. Here we collect the W-linked genes of 27 birds, covering the three major avian clades: Neoaves (songbirds), Galloanserae (chicken), and Palaeognathae (ratites and tinamous). We find that the avian W chromosomes exhibit very conserved gene content despite their independent evolution of recombination suppression. The retained W-linked genes have higher dosage-sensitive and higher expression level than the lost genes, suggesting the role of purifying selection in their retention. Moreover, they are not enriched in ancestrally female-biased genes, and have not acquired new ovary-biased expression patterns after becoming W-linked. They are broadly expressed across female tissues, and the expression profile of the W-linked genes in females is not deviated from that of the homologous Z-linked genes. Together, our new analyses suggest that female-specific positive selection on the avian W chromosomes is limited, and the gene content of the W chromosomes is mainly shaped by purifying selection.

Evolutionary Dynamics of Sex Chromosomes of Paleognathous Birds.

Standard models of sex chromosome evolution propose that recombination suppression leads to the degeneration of the heterogametic chromosome, as is seen for the Y chromosome in mammals and the W chromosome in most birds. Unlike other birds, paleognaths (ratites and tinamous) possess large nondegenerate regions on their sex chromosomes (PARs or pseudoautosomal regions). It remains unclear why these large PARs are retained over >100 Myr, and how this retention impacts the evolution of sex chromosomes within this system. To address this puzzle, we analyzed Z chromosome evolution and gene expression across 12 paleognaths, several of whose genomes have recently been sequenced. We confirm at the genomic level that most paleognaths retain large PARs. As in other birds, we find that all paleognaths have incomplete dosage compensation on the regions of the Z chromosome homologous to degenerated portions of the W (differentiated regions), but we find no evidence for enrichments of male-biased genes in PARs. We find limited evidence for increased evolutionary rates (faster-Z) either across the chromosome or in differentiated regions for most paleognaths with large PARs, but do recover signals of faster-Z evolution in tinamou species with mostly degenerated W chromosomes, similar to the pattern seen in neognaths. Unexpectedly, in some species, PAR-linked genes evolve faster on average than genes on autosomes, suggested by diverse genomic features to be due to reduced efficacy of selection in paleognath PARs. Our analysis shows that paleognath Z chromosomes are atypical at the genomic level, but the evolutionary forces maintaining largely homomorphic sex chromosomes in these species remain elusive.

Frequent intra- and inter-species introgression shapes the landscape of genetic variation in bread wheat.

BACKGROUND: Bread wheat is one of the most important and broadly studied crops. However, due to the complexity of its genome and incomplete genome collection of wild populations, the bread wheat genome landscape and domestication history remain elusive. RESULTS: By investigating the whole-genome resequencing data of 93 accessions from worldwide populations of bread wheat and its diploid and tetraploid progenitors, together with 90 published exome-capture data, we find that the B subgenome has more variations than A and D subgenomes, including SNPs and deletions. Population genetics analyses support a monophyletic origin of domesticated wheat from wild emmer in northern Levant, with substantial introgressed genomic fragments from southern Levant. Southern Levant contributes more than 676 Mb in AB subgenomes and enriched in the pericentromeric regions. The AB subgenome introgression happens at the early stage of wheat speciation and partially contributes to their greater genetic diversity. Furthermore, we detect massive alien introgressions that originated from distant species through natural and artificial hybridizations, resulting in the reintroduction of ~ 709 Mb and ~ 1577 Mb sequences into bread wheat landraces and varieties, respectively. A large fraction of these intra- and inter-introgression fragments are associated with quantitative trait loci of important traits, and selection events are also identified. CONCLUSION: We reveal the significance of multiple introgressions from distant wild populations and alien species in shaping the genetic components of bread wheat, and provide important resources and new perspectives for future wheat breeding.