Annotations of four high-quality indigenous chicken genomes identify more than one thousand missing genes in subtelomeric regions and micro-chromosomes with high G/C contents.

BACKGROUND: Although multiple chicken genomes have been assembled and annotated, the numbers of protein-coding genes in chicken genomes and their variation among breeds are still uncertain due to the low quality of these genome assemblies and limited resources used in their gene annotations. To fill these gaps, we recently assembled genomes of four indigenous chicken breeds with distinct traits at chromosome-level. In this study, we annotated genes in each of these assembled genomes using a combination of RNA-seq- and homology-based approaches. RESULTS: We identified varying numbers (17,497-17,718) of protein-coding genes in the four indigenous chicken genomes, while recovering 51 of the 274 "missing" genes in birds in general, and 36 of the 174 "missing" genes in chickens in particular. Intriguingly, based on deeply sequenced RNA-seq data collected in multiple tissues in the four breeds, we found 571 ~ 627 protein-coding genes in each genome, which were missing in the annotations of the reference chicken genomes (GRCg6a and GRCg7b/w). After removing redundancy, we ended up with a total of 1,420 newly annotated genes (NAGs). The NAGs tend to be found in subtelomeric regions of macro-chromosomes (chr1 to chr5, plus chrZ) and middle chromosomes (chr6 to chr13, plus chrW), as well as in micro-chromosomes (chr14 to chr39) and unplaced contigs, where G/C contents are high. Moreover, the NAGs have elevated quadruplexes G frequencies, while both G/C contents and quadruplexes G frequencies in their surrounding regions are also high. The NAGs showed tissue-specific expression, and we were able to verify 39 (92.9%) of 42 randomly selected ones in various tissues of the four chicken breeds using RT-qPCR experiments. Most of the NAGs were also encoded in the reference chicken genomes, thus, these genomes might harbor more genes than previously thought. CONCLUSION: The NAGs are widely distributed in wild, indigenous and commercial chickens, and they might play critical roles in chicken physiology. Counting these new genes, chicken genomes harbor more genes than originally thought.

Insight into the adaptive role of arachnid genome-wide duplication through chromosome-level genome assembly of the Western black widow spider.

Although spiders are one of the most diverse groups of arthropods, the genetic architecture of their evolutionary adaptations is largely unknown. Specifically, ancient genome-wide duplication occurring during arachnid evolution ~450 mya resulted in a vast assembly of gene families, yet the extent to which selection has shaped this variation is understudied. To aid in comparative genome sequence analyses, we provide a chromosome-level genome of the Western black widow spider (Latrodectus hesperus)-a focus due to its silk properties, venom applications, and as a model for urban adaptation. We used long-read and Hi-C sequencing data, combined with transcriptomes, to assemble 14 chromosomes in a 1.46 Gb genome, with 38,393 genes annotated, and a BUSCO score of 95.3%. Our analyses identified high repetitive gene content and heterozygosity, consistent with other spider genomes, which has led to challenges in genome characterization. Our comparative evolutionary analyses of eight genomes available for species within the Araneoidea group (orb weavers and their descendants) identified 1,827 single-copy orthologs. Of these, 155 exhibit significant positive selection primarily associated with developmental genes, and with traits linked to sensory perception. These results support the hypothesis that several traits unique to spiders emerged from the adaptive evolution of ohnologs-or retained ancestrally duplicated genes-from ancient genome-wide duplication. These comparative spider genome analyses can serve as a model to understand how positive selection continually shapes ancestral duplications in generating novel traits today within and between diverse taxonomic groups.

Entangling and disentangling mitotic chromosomes.

The DNA topological challenges generated by cellular manipulation of extremely long DNA fibers remain poorly understood. In this issue of Molecular Cell, Hildebrand et al.1 describe how mitotic chromosomes are self entangled and that disentanglement requires TOP2 activity in late mitosis.

Chromosome-scale Genome Assembly of the Rough Periwinkle Littorina saxatilis.

The intertidal gastropod Littorina saxatilis is a model system to study speciation and local adaptation. The repeated occurrence of distinct ecotypes showing different levels of genetic divergence makes L. saxatilis particularly suited to study different stages of the speciation continuum in the same lineage. A major finding is the presence of several large chromosomal inversions associated with the divergence of ecotypes and, specifically, the species offers a system to study the role of inversions in this divergence. The genome of L. saxatilis is 1.35 Gb and composed of 17 chromosomes. The first reference genome of the species was assembled using Illumina data, was highly fragmented (N50 of 44 kb), and was quite incomplete, with a BUSCO completeness of 80.1% on the Metazoan dataset. A linkage map of one full-sibling family enabled the placement of 587 Mbp of the genome into 17 linkage groups corresponding to the haploid number of chromosomes, but the fragmented nature of this reference genome limited the understanding of the interplay between divergent selection and gene flow during ecotype formation. Here, we present a newly generated reference genome that is highly contiguous, with a N50 of 67 Mb and 90.4% of the total assembly length placed in 17 super-scaffolds. It is also highly complete with a BUSCO completeness of 94.1% of the Metazoa dataset. This new reference will allow for investigations into the genomic regions implicated in ecotype formation as well as better characterization of the inversions and their role in speciation.

Chromosome-level genome provides insights into environmental adaptability and innate immunity in the common dolphin (delphinus delphis).

The common dolphin (Delphinus delphis) is widely distributed worldwide and well adapted to various habitats. Animal genomes store clues about their pasts, and can reveal the genes underlying their evolutionary success. Here, we report the first high-quality chromosome-level genome of D. delphis. The assembled genome size was 2.56 Gb with a contig N50 of 63.85 Mb. Phylogenetically, D. delphis was close to Tursiops truncatus and T. aduncus. The genome of D. delphis exhibited 428 expanded and 1,885 contracted gene families, and 120 genes were identified as positively selected. The expansion of the HSP70 gene family suggested that D. delphis has a powerful system for buffering stress, which might be associated with its broad adaptability, longevity, and detoxification capacity. The expanded IFN-α and IFN-ω gene families, as well as the positively selected genes encoding tripartite motif-containing protein 25, peptidyl-prolyl cis-trans isomerase NIMA-interacting 1, and p38 MAP kinase, were all involved in pathways for antiviral, anti-inflammatory, and antineoplastic mechanisms. The genome data also revealed dramatic fluctuations in the effective population size during the Pleistocene. Overall, the high-quality genome assembly and annotation represent significant molecular resources for ecological and evolutionary studies of Delphinus and help support their sustainable treatment and conservation.

The scales, mechanisms, and dynamics of the genome architecture.

Even when split into several chromosomes, DNA molecules that make up our genome are too long to fit into the cell nuclei unless massively folded. Such folding must accommodate the need for timely access to selected parts of the genome by transcription factors, RNA polymerases, and DNA replication machinery. Here, we review our current understanding of the genome folding inside the interphase nuclei. We consider the resulting genome architecture at three scales with a particular focus on the intermediate (meso) scale and summarize the insights gained from recent experimental observations and diverse computational models.

The chromosome-level genome assembly of the giant dobsonfly Acanthacorydalis orientalis (McLachlan, 1899).

Acanthacorydalis orientalis (McLachlan, 1899) (Megaloptera: Corydalidae) is an important freshwater-benthic invertebrate species that serves as an indicator for water-quality biomonitoring and is valuable for conservation from East Asia. Here, a high-quality reference genome for A. orientalis was constructed using Oxford Nanopore sequencing and High throughput Chromosome Conformation Capture (Hi-C) technology. The final genome size is 547.98 Mb, with the N50 values of contig and scaffold being 7.77 Mb and 50.53 Mb, respectively. The longest contig and scaffold are 20.57 Mb and 62.26 Mb in length, respectively. There are 99.75% contigs anchored onto 13 pseudo-chromosomes. Benchmarking Universal Single-Copy Orthologs (BUSCO) analysis showed that the completeness of the genome assembly is 99.01%. There are 10,977 protein-coding genes identified, of which 84.00% are functionally annotated. The genome contains 44.86% repeat sequences. This high-quality genome provides substantial data for future studies on population genetics, aquatic adaptation, and evolution of Megaloptera and other related insect groups.

Chromatin looping links gene expression to the assembly of transcription factories (Review).

Genes are not randomly dispersed within the nuclear space, instead they occupy precise sites either with respect to the nuclear lamina as well as to each other. This observation stands at the basis of the today well accepted concept of nuclear territories where any chromosome shows reproducible spatial connections with a selection of others in a general picture that meets a functional criterion where genes that answer the same stimuli are grouped in the same sites. In fact, transcription is not visible widely dispersed throughout the nucleus but is gathered in several 'granules', called transcription factories that accommodates ~10 genes concurrently transcribed. This dynamic behavior of chromosomes is allowed by changes in chromatin plasticity that are governed by several classes of proteins that either modify its building or induce post­translational modifications in the protein component of nucleosomes, triggering formation of chromosome loops that modify the location of specific sites along the DNA strand. For example, transcription associated to nuclear receptors benefits of the generation of nuclear ROS that induce nicks following activation of the DNA repair apparatus that enhance helix unfolding and chromosome bridging. In the present review, the role that protocols facing elucidation of chromosome architecture are playing and will play in the near future were highlighted in order to investigate composition of the transcription factories assembled in response of a specific trigger: The estrogen­sensitive transcription was cited but the authors are convinced that the same portrait will be observed with a multitude of (if not all) other stimuli.

High-Quality, Chromosome-Level Reference Genomes of the Viviparous Caribbean Skinks Spondylurus nitidus and S. culebrae.

New World mabuyine skinks are a diverse radiation of morphologically cryptic lizards with unique reproductive biologies. Recent studies examining population-level data (morphological, ecological, and genomic) have uncovered novel biodiversity and phenotypes, including the description of dozens of new species and insights into the evolution of their highly complex placental structures. Beyond the potential for this diverse group to serve as a model for the evolution of viviparity in lizards, much of the taxonomic diversity is concentrated in regions experiencing increasing environmental instability from climate and anthropogenic change. Consequently, a better understanding of genome structure and diversity will be an important tool in the adaptive management and conservation of this group. Skinks endemic to Caribbean islands are particularly vulnerable to global change with several species already considered likely extinct and several remaining species either endangered or threatened. Combining PacBio long-read sequencing, Hi-C, and RNAseq data, here we present the first genomic resources for this group by describing new chromosome-level reference genomes for the Puerto Rican Skink Spondylurus nitidus and the Culebra Skink S. culebrae. Results indicate two high quality genomes, both ∼1.4 Gb, assembled nearly telomere to telomere with complete mitochondrion assembly and annotation.

A working model for the formation of Robertsonian chromosomes.

Robertsonian chromosomes form by fusion of two chromosomes that have centromeres located near their ends, known as acrocentric or telocentric chromosomes. This fusion creates a new metacentric chromosome and is a major mechanism of karyotype evolution and speciation. Robertsonian chromosomes are common in nature and were first described in grasshoppers by the zoologist W. R. B. Robertson more than 100 years ago. They have since been observed in many species, including catfish, sheep, butterflies, bats, bovids, rodents and humans, and are the most common chromosomal change in mammals. Robertsonian translocations are particularly rampant in the house mouse, Mus musculus domesticus, where they exhibit meiotic drive and create reproductive isolation. Recent progress has been made in understanding how Robertsonian chromosomes form in the human genome, highlighting some of the fundamental principles of how and why these types of fusion events occur so frequently. Consequences of these fusions include infertility and Down's syndrome. In this Hypothesis, I postulate that the conditions that allow these fusions to form are threefold: (1) sequence homology on non-homologous chromosomes, often in the form of repetitive DNA; (2) recombination initiation during meiosis; and (3) physical proximity of the homologous sequences in three-dimensional space. This Hypothesis highlights the latest progress in understanding human Robertsonian translocations within the context of the broader literature on Robertsonian chromosomes.

Evidence of Nonrandom Patterns of Functional Chromosome Organization in Danaus plexippus.

Our understanding on the interplay between gene functionality and gene arrangement at different chromosome scales relies on a few Diptera and the honeybee, species with quality reference genome assemblies, accurate gene annotations, and abundant transcriptome data. Using recently generated 'omic resources in the monarch butterfly Danaus plexippus, a species with many more and smaller chromosomes relative to Drosophila species and the honeybee, we examined the organization of genes preferentially expressed at broadly defined developmental stages (larva, pupa, adult males, and adult females) at both fine and whole-chromosome scales. We found that developmental stage-regulated genes do not form more clusters, but do form larger clusters, than expected by chance, a pattern consistent across the gene categories examined. Notably, out of the 30 chromosomes in the monarch genome, 12 of them, plus the fraction of the chromosome Z that corresponds to the ancestral Z in other Lepidoptera, were found enriched for developmental stage-regulated genes. These two levels of nonrandom gene organization are not independent as enriched chromosomes for developmental stage-regulated genes tend to harbor disproportionately large clusters of these genes. Further, although paralogous genes were overrepresented in gene clusters, their presence is not enough to explain two-thirds of the documented cases of whole-chromosome enrichment. The composition of the largest clusters often included paralogs from more than one multigene family as well as unrelated single-copy genes. Our results reveal intriguing patterns at the whole-chromosome scale in D. plexippus while shedding light on the interplay between gene expression and chromosome organization beyond Diptera and Hymenoptera.

A chromosome-level genome assembly of the pig-nosed turtle (Carettochelys insculpta).

The pig-nosed turtle (Carettochelys insculpta) represents the only extant species within the Carettochelyidae family, is a unique Trionychia member fully adapted to aquatic life and currently facing endangerment. To enhance our understanding of this species and contribute to its conservation efforts, we employed high-fidelity (HiFi) and Hi-C sequencing technology to generate its genome assembly at the chromosome level. The assembly result spans 2.18 Gb, with a contig N50 of 126 Mb, encompassing 34 chromosomes that account for 99.6% of the genome. The assembly has a BUSCO score above 95% with different databases and strong collinearity with Yangtze giant softshell turtles (Rafetus swinhoei), indicating its completeness and continuity. A total of 19,175 genes and 46.86% repetitive sequences were annotated. The availability of this chromosome-scale genome represents a valuable resource for the pig-nosed turtle, providing insights into its aquatic adaptation and serving as a foundation for future turtle research.

Footprints of loop extrusion in statistics of intra-chromosomal distances: An analytically solvable model.

Active loop extrusion-the process of formation of dynamically growing chromatin loops due to the motor activity of DNA-binding protein complexes-is a firmly established mechanism responsible for chromatin spatial organization at different stages of a cell cycle in eukaryotes and bacteria. The theoretical insight into the effect of loop extrusion on the experimentally measured statistics of chromatin conformation can be gained with an appropriately chosen polymer model. Here, we consider the simplest analytically solvable model of an interphase chromosome, which is treated as an ideal chain with disorder of sufficiently sparse random loops whose conformations are sampled from the equilibrium ensemble. This framework allows us to arrive at the closed-form analytical expression for the mean-squared distance between pairs of genomic loci, which is valid beyond the one-loop approximation in diagrammatic representation. In addition, we analyze the loop-induced deviation of chain conformations from the Gaussian statistics by calculating kurtosis of probability density of the pairwise separation vector. The presented results suggest the possible ways of estimating the characteristics of the loop extrusion process based on the experimental data on the scale-dependent statistics of intra-chromosomal pair-wise distances.

A chromosome-level genome assembly of East Asia endemic minnow Zacco platypus.

Zacco platypus is an endemic colorful freshwater minnow that is intensively distributed in East Asia. In this study, two adult female individuals collected from Haihe River basin were used for karyotypic study and genome sequencing, respectively. The karyotype formula of Z. platypus is 2N = 48 = 18 M + 24SM/ST + 6 T. We used PacBio long-read sequencing and Hi-C technology to assemble a chromosome-level genome of Z. platypus. As a result, an 814.87 Mb genome was assembled with the PacBio long reads. Subsequently, 98.64% assembled sequences were anchored into 24 chromosomes based on the Hi-C data. The chromosome-level assembly contained 54 scaffolds with a N50 length of 32.32 Mb. Repeat elements accounted for 52.35% in genome, and 24,779 protein-coding genes were predicted, with 92.11% were functionally annotated with the public databases. BUSCO analysis yielded a completeness score of 96.5%. This high-quality genome assembly provides valuable resources for future functional genomic research, comparative genomics, and evolutionary studies of genus Zacco.

Chromosome-Level Assembly and Annotation of the Pearly Heath Coenonympha arcania Butterfly Genome.

We present the first chromosome-level genome assembly and annotation of the pearly heath Coenonympha arcania, generated with a PacBio HiFi sequencing approach and complemented with Hi-C data. We additionally compare synteny, gene, and repeat content between C. arcania and other Lepidopteran genomes. This reference genome will enable future population genomics studies with Coenonympha butterflies, a species-rich genus that encompasses some of the most highly endangered butterfly taxa in Europe.

Mitotic chromosomes are self-entangled and disentangle through a topoisomerase-II-dependent two-stage exit from mitosis.

The topological state of chromosomes determines their mechanical properties, dynamics, and function. Recent work indicated that interphase chromosomes are largely free of entanglements. Here, we use Hi-C, polymer simulations, and multi-contact 3C and find that, by contrast, mitotic chromosomes are self-entangled. We explore how a mitotic self-entangled state is converted into an unentangled interphase state during mitotic exit. Most mitotic entanglements are removed during anaphase/telophase, with remaining ones removed during early G1, in a topoisomerase-II-dependent process. Polymer models suggest a two-stage disentanglement pathway: first, decondensation of mitotic chromosomes with remaining condensin loops produces entropic forces that bias topoisomerase II activity toward decatenation. At the second stage, the loops are released, and the formation of new entanglements is prevented by lower topoisomerase II activity, allowing the establishment of unentangled and territorial G1 chromosomes. When mitotic entanglements are not removed in experiments and models, a normal interphase state cannot be acquired.

Multiplex DNA fluorescence in situ hybridization to analyze maternal vs. paternal C. elegans chromosomes.

Recent advances in microscopy have enabled studying chromosome organization at the single-molecule level, yet little is known about inherited chromosome organization. Here we adapt single-molecule chromosome tracing to distinguish two C. elegans strains (N2 and HI) and find that while their organization is similar, the N2 chromosome influences the folding parameters of the HI chromosome, in particular the step size, across generations. Furthermore, homologous chromosomes overlap frequently, but alignment between homologous regions is rare, suggesting that transvection is unlikely. We present a powerful tool to investigate chromosome architecture and to track the parent of origin.

A high-quality chromosome-level genome assembly of the Chinese medaka Oryzias sinensis.

Oryzias sinensis, also known as Chinese medaka or Chinese ricefish, is a commonly used animal model for aquatic environmental assessment in the wild as well as gene function validation or toxicology research in the lab. Here, a high-quality chromosome-level genome assembly of O. sinensis was generated using single-tube long fragment read (stLFR) reads, Nanopore long-reads, and Hi-C sequencing data. The genome is 796.58 Mb, and a total of 712.17 Mb of the assembled sequences were anchored to 23 pseudo-chromosomes. A final set of 22,461 genes were annotated, with 98.67% being functionally annotated. The Benchmarking Universal Single-Copy Orthologs (BUSCO) benchmark of genome assembly and gene annotation reached 95.1% (93.3% single-copy) and 94.6% (91.7% single-copy), respectively. Furthermore, we also use ATAC-seq to uncover chromosome transposase-accessibility as well as related genome area function enrichment for Oryzias sinensis. This study offers a new improved foundation for future genomics research in Chinese medaka.

Haplotype-resolved 3D chromatin architecture of the hybrid pig.

In diploid mammals, allele-specific three-dimensional (3D) genome architecture may lead to imbalanced gene expression. Through ultradeep in situ Hi-C sequencing of three representative somatic tissues (liver, skeletal muscle, and brain) from hybrid pigs generated by reciprocal crosses of phenotypically and physiologically divergent Berkshire and Tibetan pigs, we uncover extensive chromatin reorganization between homologous chromosomes across multiple scales. Haplotype-based interrogation of multi-omic data revealed the tissue dependence of 3D chromatin conformation, suggesting that parent-of-origin-specific conformation may drive gene imprinting. We quantify the effects of genetic variations and histone modifications on allelic differences of long-range promoter-enhancer contacts, which likely contribute to the phenotypic differences between the parental pig breeds. We also observe the fine structure of somatically paired homologous chromosomes in the pig genome, which has a functional implication genome-wide. This work illustrates how allele-specific chromatin architecture facilitates concomitant shifts in allele-biased gene expression, as well as the possible consequential phenotypic changes in mammals.