A chromosome-scale genome assembly of Malus domestica,a multi-stress resistant apple variety

Hanfu apple is the main cultivar grown in the cool areas of Northeast, Northwest, and North China. Here, we proposed a chromosome-level Hanfu genome assembly using PacBio, Illumina and Hi-C sequencing data. The total contig length was 628.99 Mb, with scaffold and contig N50 sizes of 36.18 Mb and 1.25 Mb, respectively. The Hanfu genome had a total of 39,617 genes, of which we predicted the function for 38,816. Evolutionary analysis showed that Hanfu may have undergone a γ-event, a recent whole-genome duplication. A comparative analysis was conducted on the genomes of Hanfu and homozygous triploid HFTH1, which were cultured using the anthers of diploid Hanfu apples. Three variants were identified, including 2,155,184 single nucleotide polymorphisms (SNPs), 413,108 insertions/deletions (indels), and 7,587 structural variants (SVs).This high-quality genome will provide a reference for the genetic improvement of apples and the breeding of more varieties with high resistance and high quality.     

Improved genome assembly of Chinese shrimp (Fenneropenaeus chinensis) suggests adaptation to the environment during evolution and domestication

A high-quality reference genome is necessary to determine the molecular mechanisms underlying important biological phenomena; therefore, in the present study, a chromosome-level genome assembly of the Chinese shrimp Fenneropenaeus chinensis was performed. Muscle of a male shrimp was sequenced using PacBio platform, and assembled by Hi-C technology. The assembled F. chinensis genome was 1.47 Gb with contig N50 of 472.84 Kb, including 57.73% repetitive sequences, and was anchored to 43 pseudochromosomes, with scaffold N50 of 36.87 Mb. In total, 25,026 protein-coding genes were predicted. The genome size of F. chinensis showed significant contraction in comparison with that of other penaeid species, which is likely related to migration observed in this species. However, the F. chinensis genome included several expanded gene families related to cellular processes and metabolic processes, and the contracted gene families were associated with virus infection process. The findings signify the adaptation of F. chinensis to the selection pressure of migration and cold environment. Furthermore, the selection signature analysis identified genes associated with metabolism, phototransduction, and nervous system in cultured shrimps when compared with wild population, indicating targeted, artificial selection of growth, vision, and behavior during domestication. The construction of the genome of F. chinensis provided valuable information for the further genetic mechanism analysis of important biological processes, and will facilitate the research of genetic changes during evolution.     

The diploid genome sequence of an individual human

Presented here is a genome sequence of an individual human. It was produced from ∼32 million random DNA fragments, sequenced by Sanger dideoxy technology and assembled into 4,528 scaffolds, comprising 2,810 million bases (Mb) of contiguous sequence with approximately 7.5-fold coverage for any given region. We developed a modified version of the Celera assembler to facilitate the identification and comparison of alternate alleles within this individual diploid genome. Comparison of this genome and the National Center for Biotechnology Information human reference assembly revealed more than 4.1 million DNA variants, encompassing 12.3 Mb. These variants (of which 1,288,319 were novel) included 3,213,401 single nucleotide polymorphisms (SNPs), 53,823 block substitutions (2–206 bp), 292,102 heterozygous insertion/deletion events (indels)(1–571 bp), 559,473 homozygous indels (1–82,711 bp), 90 inversions, as well as numerous segmental duplications and copy number variation regions. Non-SNP DNA variation accounts for 22% of all events identified in the donor, however they involve 74% of all variant bases. This suggests an important role for non-SNP genetic alterations in defining the diploid genome structure. Moreover, 44% of genes were heterozygous for one or more variants. Using a novel haplotype assembly strategy, we were able to span 1.5 Gb of genome sequence in segments >200 kb, providing further precision to the diploid nature of the genome. These data depict a definitive molecular portrait of a diploid human genome that provides a starting point for future genome comparisons and enables an era of individualized genomic information.     

Chromosomal‐level assembly of Takifugu obscurus (Abe, 1949) genome using third‐generation DNA sequencing and Hi‐C analysis

The Tetraodontidae family are known to have relatively small and compact genomes compared to other vertebrates. The obscure puffer fish Takifugu obscurus is an anadromous species that migrates to freshwater from the sea for spawning. Thus the euryhaline characteristics of T. obscurus have been investigated to gain understanding of their survival ability, osmoregulation, and other homeostatic mechanisms in both freshwater and seawater. In this study, a high quality chromosome‐level reference genome for T. obscurus was constructed using long‐read Pacific Biosciences (PacBio) Sequel sequencing and a Hi‐C‐based chromatin contact map platform. The final genome assembly of T. obscurus is 381 Mb, with a contig N50 length of 3,296 kb and longest length of 10.7 Mb, from a total of 62 Gb of raw reads generated using single‐molecule real‐time sequencing technology from a PacBio Sequel platform. The PacBio data were further clustered into chromosome‐scale scaffolds using a Hi‐C approach, resulting in a 373 Mb genome assembly with a contig N50 length of 15.2 Mb and and longest length of 28 Mb. When we directly compared the 22 longest scaffolds of T. obscurus to the 22 chromosomes of the tiger puffer Takifugu rubripes, a clear one‐to‐one orthologous relationship was observed between the two species, supporting the chromosome‐level assembly of T. obscurus. This genome assembly can serve as a valuable genetic resource for exploring fugu‐specific compact genome characteristics, and will provide essential genomic information for understanding molecular adaptations to salinity fluctuations and the evolution of osmoregulatory mechanisms.     

A high-quality genome of the dobsonfly Neoneuromus ignobilis reveals molecular convergences in aquatic insects

Neoneuromus ignobilis is an archaic holometabolous aquatic predatory insect. However, a lack of genomic resources hinders the use of whole genome sequencing to explore their genetic basis and molecular mechanisms for adaptive evolution. Here, we provided a high-contiguity, chromosome-level genome assembly of N. ignobilis using high coverage Nanopore and PacBio reads with the Hi-C technique. The final assembly is 480.67 MB in size, containing 12 telomere-ended pseudochromosomes with only 17 gaps. We compared 42 hexapod species genomes including six independent lineages comprising 11 aquatic insects, and found convergent expansions of long wavelength-sensitive and blue-sensitive opsins, thermal stress response TRP channels, and sulfotransferases in aquatic insects, which may be related to their aquatic adaptation. We also detected strong nonrandom signals of convergent amino acid substitutions in aquatic insects. Collectively, our comparative genomic analysis revealed the evidence of molecular convergences in aquatic insects during both gene family evolution and convergent amino acid substitutions.     

Using de novo assembly to identify structural variation of eight complex immune system gene regions

Driven by the necessity to survive environmental pathogens, the human immune system has evolved exceptional diversity and plasticity, to which several factors contribute including inheritable structural polymorphism of the underlying genes. Characterizing this variation is challenging due to the complexity of these loci, which contain extensive regions of paralogy, segmental duplication and high copy-number repeats, but recent progress in long-read sequencing and optical mapping techniques suggests this problem may now be tractable. Here we assess this by using long-read sequencing platforms from PacBio and Oxford Nanopore, supplemented with short-read sequencing and Bionano optical mapping, to sequence DNA extracted from CD14⁺ monocytes and peripheral blood mononuclear cells from a single European individual identified as HV31. We use this data to build a de novo assembly of eight genomic regions encoding four key components of the immune system, namely the human leukocyte antigen, immunoglobulins, T cell receptors, and killer-cell immunoglobulin-like receptors. Validation of our assembly using k-mer based and alignment approaches suggests that it has high accuracy, with estimated base-level error rates below 1 in 10 kb, although we identify a small number of remaining structural errors. We use the assembly to identify heterozygous and homozygous structural variation in comparison to GRCh38. Despite analyzing only a single individual, we find multiple large structural variants affecting core genes at all three immunoglobulin regions and at two of the three T cell receptor regions. Several of these variants are not accurately callable using current algorithms, implying that further methodological improvements are needed. Our results demonstrate that assessing haplotype variation in these regions is possible given sufficiently accurate long-read and associated data. Continued reductions in the cost of these technologies will enable application of these methods to larger samples and provide a broader catalogue of germline structural variation at these loci, an important step toward making these regions accessible to large-scale genetic association studies.     

Towards a whole‐genome sequence for rye (Secale cereale L.)

We report on a whole‐genome draft sequence of rye (Secale cereale L.). Rye is a diploid Triticeae species closely related to wheat and barley, and an important crop for food and feed in Central and Eastern Europe. Through whole‐genome shotgun sequencing of the 7.9‐Gbp genome of the winter rye inbred line Lo7 we obtained a de novo assembly represented by 1.29 million scaffolds covering a total length of 2.8 Gbp. Our reference sequence represents nearly the entire low‐copy portion of the rye genome. This genome assembly was used to predict 27 784 rye gene models based on homology to sequenced grass genomes. Through resequencing of 10 rye inbred lines and one accession of the wild relative S. vavilovii, we discovered more than 90 million single nucleotide variants and short insertions/deletions in the rye genome. From these variants, we developed the high‐density Rye600k genotyping array with 600 843 markers, which enabled anchoring the sequence contigs along a high‐density genetic map and establishing a synteny‐based virtual gene order. Genotyping data were used to characterize the diversity of rye breeding pools and genetic resources, and to obtain a genome‐wide map of selection signals differentiating the divergent gene pools. This rye whole‐genome sequence closes a gap in Triticeae genome research, and will be highly valuable for comparative genomics, functional studies and genome‐based breeding in rye.     

Copy number increases of transposable elements and protein‐coding genes in an invasive fish of hybrid origin

Evolutionary dynamics of structural genetic variation in lineages of hybrid origin is not well explored, although structural mutations may increase in controlled hybrid crosses. We therefore tested whether structural variants accumulate in a fish of recent hybrid origin, invasive Cottus, relative to both parental species Cottus rhenanus and Cottus perifretum. Copy‐number variation in exons of 10,979 genes was assessed using comparative genome hybridization arrays. Twelve genes showed significantly higher copy numbers in invasive Cottus compared to both parents. This coincided with increased expression for three genes related to vision, detoxification and muscle development, suggesting possible gene dosage effects. Copy number increases of putative transposons were assessed by comparative mapping of genomic DNA reads against a de novo assembly of 1,005 repetitive elements. In contrast to exons, copy number increases of repetitive elements were common (20.7%) in invasive Cottus, whereas decrease was very rare (0.01%). Among the increased repetitive elements, 53.8% occurred at higher numbers in C. perifretum compared to C. rhenanus, while only 1.4% were more abundant in C. rhenanus. This implies a biased mutational process that amplifies genetic material from one ancestor. To assess the frequency of de novo mutations through hybridization, we screened 64 laboratory‐bred F₂ offspring between the parental species for copy‐number changes at five candidate loci. We found no evidence for new structural variants, indicating that they are too rare to be detected given our sampling scheme. Instead, they must have accumulated over more generations than we observed in a controlled cross.     

Genome‐wide characterization and expression analysis of genetic variants in sweet orange

Heterozyosity is an important feature of many plant genomes, and is related to heterosis. Sweet orange, a highly heterozygous species, is thought to have originated from an inter‐species hybrid between pummelo and mandarin. To investigate the heterozygosity of the sweet orange genome and examine how this heterozygosity affects gene expression, we characterized the genome of Valencia orange for single nucleotide variations (SNVs), small insertions and deletions (InDels) and structural variations (SVs), and determined their functional effects on protein‐coding genes and non‐coding sequences. Almost half of the genes containing large‐effect SNVs and InDels were expressed in a tissue‐specific manner. We identified 3542 large SVs (>50 bp), including deletions, insertions and inversions. Most of the 296 genes located in large‐deletion regions showed low expression levels. RNA‐Seq reads and DNA sequencing reads revealed that the alleles of 1062 genes were differentially expressed. In addition, we detected approximately 42 Mb of contigs that were not found in the reference genome of a haploid sweet orange by de novo assembly of unmapped reads, and annotated 134 protein‐coding genes within these contigs. We discuss how this heterozygosity affects the quality of genome assembly. This study advances our understanding of the genome architecture of sweet orange, and provides a global view of gene expression at heterozygous loci.     

A de novo chromosome‐level genome assembly of Coregonus sp. “Balchen”: One representative of the Swiss Alpine whitefish radiation

Salmonids are of particular interest to evolutionary biologists due to their incredible diversity of life‐history strategies and the speed at which many salmonid species have diversified. In Switzerland alone, over 30 species of Alpine whitefish from the subfamily Coregoninae have evolved since the last glacial maximum, with species exhibiting a diverse range of morphological and behavioural phenotypes. This, combined with the whole genome duplication which occurred in the ancestor of all salmonids, makes the Alpine whitefish radiation a particularly interesting system in which to study the genetic basis of adaptation and speciation and the impacts of ploidy changes and subsequent rediploidization on genome evolution. Although well‐curated genome assemblies exist for many species within Salmonidae, genomic resources for the subfamily Coregoninae are lacking. To assemble a whitefish reference genome, we carried out PacBio sequencing from one wild‐caught Coregonus sp. “Balchen” from Lake Thun to ~90× coverage. PacBio reads were assembled independently using three different assemblers, falcon , canu and wtdbg2 and subsequently scaffolded with additional Hi‐C data. All three assemblies were highly contiguous, had strong synteny to a previously published Coregonus linkage map, and when mapping additional short‐read data to each of the assemblies, coverage was fairly even across most chromosome‐scale scaffolds. Here, we present the first de novo genome assembly for the Salmonid subfamily Coregoninae. The final 2.2‐Gb wtdbg2 assembly included 40 scaffolds, an N50 of 51.9 Mb and was 93.3% complete for BUSCOs. The assembly consisted of ~52% transposable elements and contained 44,525 genes.     

De novo sequencing and chromosomal‐scale genome assembly of leopard coral grouper,Plectropomus leopardus

The leopard coral grouper, Plectropomus leopardus, belonging to the family Epinephelinae, is a carnivorous coral reef fish widely distributed in tropical and subtropical waters of the Indo‐Pacific. Due to its appealing body appearance and delicious taste, P. leopardus has become a popular commercial fish for aquaculture in many countries. However, the lack of genomic and molecular resources for P. leopardus has hindered study of its biology and genomic breeding programmes. Here we report the de novo sequencing and assembly of the P. leopardus genome using a combination of 10 × Genomics, high‐throughput chromosome conformation capture (Hi‐C) and PacBio long‐read sequencing technologies. The genome assembly has a total length of 881.55 Mb with a scaffold N50 of 34.15 Mb, consisting of 24 pseudochromosome scaffolds. busco analysis showed that 97.2% of the conserved single‐copy genes were retrieved, indicating the assembly was almost entire. We predicted 25,248 protein‐coding genes, among which 96.5% were functionally annotated. Comparative genomic analyses revealed that gene family expansions in P. leopardus were associated with immune‐related pathways. In addition, we identified 5,178,453 single nucleotide polymorphisms based on genome resequencing of 54 individuals. The P. leopardus genome and genomic variation data provide valuable genomic resources for studies of its genetics, evolution and biology. In particular, it is expected to benefit the development of genomic breeding programmes in the farming industry.     

A chromosome-level reference genome of red swamp crayfish Procambarus clarkii provides insights into the gene families regarding growth or development in crustaceans

Red swamp crayfish Procambarus clarkii is an ecologically and economically important crustacean species. Here, based on a de novo assembly strategy combining PacBio with Hi-C sequencing, we presented a high quality chromosome-level P. clarkii genome. The assembled genome is 2.75 Gb in size with a contig N50 of 216.75 kb. Transposable elements (TEs) make up the largest fraction of the genome (~79.61%), and LINEs comprise the majority of the TEs. Frequent molting and rapid growth of the red swamp crayfish may be explained by the expansion of multiple gene families regarding growth or development. Phylogenetic analysis revealed that P. clarkii diverged from Portunus trituberculatus at 278–407 million years ago (Mya). PSMC analysis identified multiple bottleneck events of the P. clarkii population between 2 kaBP to 14 kaBP. The obtained P. clarkii genome should not only facilitate us understanding the development and evolution of the crayfish species, but also contribute to the genetic improvement in future breeding selections.     

Genome of an iconic Australian bird: High‐quality assembly and linkage map of the superb fairy‐wren (Malurus cyaneus)

The superb fairy‐wren, Malurus cyaneus, is one of the most iconic Australian passerine species. This species belongs to an endemic Australasian clade, Meliphagides, which diversified early in the evolution of the oscine passerines. Today, the oscine passerines comprise almost half of all avian species diversity. Despite the rapid increase of available bird genome assemblies, this part of the avian tree has not yet been represented by a high‐quality reference. To rectify that, we present the first high‐quality genome assembly of a Meliphagides representative: the superb fairy‐wren. We combined Illumina shotgun and mate‐pair sequences, PacBio long‐reads, and a genetic linkage map from an intensively sampled pedigree of a wild population to generate this genome assembly. Of the final assembled 1.07‐Gb genome, 975 Mb (90.4%) was anchored onto 25 pseudochromosomes resulting in a final superscaffold N50 of 68.11 Mb. This high‐quality bird genome assembly is one of only a handful which is also accompanied by a genetic map and recombination landscape. In comparison to other pedigree‐based bird genetic maps, we find that the fairy‐wren genetic map more closely resembles those of Taeniopygia guttata and Parus major maps, unlike the Ficedula albicollis map which more closely resembles that of Gallus gallus. Lastly, we also provide a predictive gene and repeat annotation of the genome assembly. This new high‐quality, annotated genome assembly will be an invaluable resource not only regarding the superb fairy‐wren species and relatives but also broadly across the avian tree by providing a novel reference point for comparative genomic analyses.     

Construction and comparison of three reference‐quality genome assemblies for soybean

We report reference‐quality genome assemblies and annotations for two accessions of soybean (Glycine max) and for one accession of Glycine soja, the closest wild relative of G. max. The G. max assemblies provided are for widely used US cultivars: the northern line Williams 82 (Wm82) and the southern line Lee. The Wm82 assembly improves the prior published assembly, and the Lee and G. soja assemblies are new for these accessions. Comparisons among the three accessions show generally high structural conservation, but nucleotide difference of 1.7 single‐nucleotide polymorphisms (snps) per kb between Wm82 and Lee, and 4.7 snps per kb between these lines and G. soja. snp distributions and comparisons with genotypes of the Lee and Wm82 parents highlight patterns of introgression and haplotype structure. Comparisons against the US germplasm collection show placement of the sequenced accessions relative to global soybean diversity. Analysis of a pan‐gene collection shows generally high conservation, with variation occurring primarily in genomically clustered gene families. We found approximately 40–42 inversions per chromosome between either Lee or Wm82v4 and G. soja, and approximately 32 inversions per chromosome between Wm82 and Lee. We also investigated five domestication loci. For each locus, we found two different alleles with functional differences between G. soja and the two domesticated accessions. The genome assemblies for multiple cultivated accessions and for the closest wild ancestor of soybean provides a valuable set of resources for identifying causal variants that underlie traits for the domestication and improvement of soybean, serving as a basis for future research and crop improvement efforts for this important crop species.     

Rare and common regulatory variation in population-scale sequenced human genomes

Population-scale genome sequencing allows the characterization of functional effects of a broad spectrum of genetic variants underlying human phenotypic variation. Here, we investigate the influence of rare and common genetic variants on gene expression patterns, using variants identified from sequencing data from the 1000 genomes project in an African and European population sample and gene expression data from lymphoblastoid cell lines. We detect comparable numbers of expression quantitative trait loci (eQTLs) when compared to genotypes obtained from HapMap 3, but as many as 80% of the top expression quantitative trait variants (eQTVs) discovered from 1000 genomes data are novel. The properties of the newly discovered variants suggest that mapping common causal regulatory variants is challenging even with full resequencing data; however, we observe significant enrichment of regulatory effects in splice-site and nonsense variants. Using RNA sequencing data, we show that 46.2% of nonsynonymous variants are differentially expressed in at least one individual in our sample, creating widespread potential for interactions between functional protein-coding and regulatory variants. We also use allele-specific expression to identify putative rare causal regulatory variants. Furthermore, we demonstrate that outlier expression values can be due to rare variant effects, and we approximate the number of such effects harboured in an individual by effect size. Our results demonstrate that integration of genomic and RNA sequencing analyses allows for the joint assessment of genome sequence and genome function.