Chromosome‐level genome assembly of a cyprinid fish Onychostoma macrolepis by integration of nanopore sequencing,Bionano and Hi‐C technology

Onychostoma macrolepis is an emerging commercial cyprinid fish species. It is a model system for studies of sexual dimorphism and genome evolution. Here, we report the chromosome‐level assembly of the O.macrolepis genome obtained from the integration of nanopore long‐read sequencing with physical maps produced using Bionano and Hi‐C technology. A total of 87.9 Gb of nanopore sequence provided approximately 100‐fold coverage of the genome. The preliminary genome assembly was 883.2 Mb in size with a contig N50 size of 11.2 Mb. The 969 corrected contigs obtained from Bionano optical mapping were assembled into 853 scaffolds and produced an assembly of 886.5 Mb with a scaffold N50 of 16.5 Mb. Finally, using the Hi‐C data, 881.3 Mb (99.4% of genome) in 526 scaffolds were anchored and oriented in 25 chromosomes ranging in size from 25.27 to 56.49 Mb. In total, 24,770 protein‐coding genes were predicted in the genome, and ~96.85% of the genes were functionally annotated. The annotated assembly contains 93.3% complete genes from the BUSCO reference set. In addition, we identified 409 Mb (46.23% of the genome) of repetitive sequence, and 11,213 non‐coding RNAs, in the genome. Evolutionary analysis revealed that O. macrolepis diverged from common carp approximately 24.25 million years ago. The chromosomes of O. macrolepis showed an unambiguous correspondence to the chromosomes of zebrafish. The high‐quality genome assembled in this work provides a valuable genomic resource for further biological and evolutionary studies of O. macrolepis.     

Chromosome‐level genome assembly of the greenhouse whitefly (Trialeurodes vaporariorum Westwood)

The greenhouse whitefly, Trialeurodes vaporariorum Westwood, is an agricultural pest of global importance. Here we report a 787‐Mb high‐quality draft genome sequence of T. vaporariorum assembled from PacBio long reads and Hi‐C chromatin interaction maps, which has scaffold and contig N50 lengths of 70 Mb and 500 kb, respectively, and contains 18,275 protein‐coding genes. About 98.8% of the assembled contigs were placed onto the 11 T. vaporariorum chromosomes. Comparative genomic analysis reveals significantly expanded gene families such as aspartyl proteases in T. vaporariorum compared to Bemisia tabaci Mediterranean (MED) and Middle East‐Asia Minor 1 (MEAM1). Furthermore, the cytochrome CYP6 subfamily shows significant expansion in T. vaporariorum and several genes in this subfamily display developmental stage‐specific expression patterns. The high‐quality T. vaporariorum genome provides a valuable resource for research in a broad range of areas such as fundamental molecular ecology, insect–plant/insect–microorganism or virus interactions and pest resistance management.     

Sequence‐tagged high‐density genetic maps of Zoysia japonica provide insights into genome evolution in Chloridoideae

Zoysiagrass (Zoysia spp.), belonging to the genus Zoysia in the subfamily Chloridoideae, is widely used in domestic lawns, sports fields and as forage. We constructed high‐density genetic maps of Zoysia japonica using a restriction site‐associated DNA sequencing (RAD‐Seq) approach and an F₁ mapping population derived from a cross between ‘Carrizo’ and ‘El Toro’. Two linkage maps were constructed, one for each of the parents. A map consisting of 2408 RAD markers distributed on 21 linkage groups was constructed for ‘Carrizo’. Another map with 1230 RAD markers mapped on 20 linkage groups was constructed for ‘El Toro’. The average distance between adjacent markers of the two maps was at 0.56 and 1.4 cM, respectively. Comparative genomics analysis was carried out among zoysiagrass, rice and sorghum genomes and a highly conserved collinearity in the gene order was observed among the three genomes. Chromosome collinearity was disrupted at centromeric regions for each chromosome pair between zoysiagrass and sorghum genomes. However, no obvious synteny gaps were observed across the centromeric regions between zoysiagrass and rice genomes. Two homologous chromosomes for each of the 10 sorghum chromosomes were found in the zoysiagrass genome, indicating an allotetraploid origin for zoysiagrass. The reduction of the basic chromosome number from 12 to 10 in chloridoids and panicoids took place via independent single‐step nested chromosome fusion events after the two subfamilies diverged from a common ancestor. The genetic maps will assist in genome sequence assembly, targeted gene isolation and comparative genomic analyses among grasses.     

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 chromosome‐level genome assembly of a generalist herbivore,Trichoplusia ni

The cabbage looper, Trichoplusia ni, is a globally distributed highly polyphagous herbivore and an important agricultural pest. T. ni has evolved resistance to various chemical insecticides, and is one of the only two insect species that have evolved resistance to the biopesticide Bacillus thuringiensis (Bt) in agricultural systems and has been selected for resistance to baculovirus infections. We report a 333‐Mb high‐quality T. ni genome assembly, which has N50 lengths of scaffolds and contigs of 4.6 Mb and 140 Kb, respectively, and contains 14,384 protein‐coding genes. High‐density genetic maps were constructed to anchor 305 Mb (91.7%) of the assembly to 31 chromosomes. Comparative genomic analysis of T. ni with Bombyx mori showed enrichment of tandemly duplicated genes in T. ni in families involved in detoxification and digestion, consistent with the broad host range of T. ni. High levels of genome synteny were found between T. ni and other sequenced lepidopterans. However, genome synteny analysis of T. ni and the T. ni derived cell line High Five (Hi5) indicated extensive genome rearrangements in the cell line. These results provided the first genomic evidence revealing the high instability of chromosomes in lepidopteran cell lines known from karyotypic observations. The high‐quality T. ni genome sequence provides a valuable resource for research in a broad range of areas including fundamental insect biology, insect‐plant interactions and co‐evolution, mechanisms and evolution of insect resistance to chemical and biological pesticides, and technology development for insect pest management.     

A high‐density linkage map enables a second‐generation collared flycatcher genome assembly and reveals the patterns of avian recombination rate variation and chromosomal evolution

Detailed linkage and recombination rate maps are necessary to use the full potential of genome sequencing and population genomic analyses. We used a custom collared flycatcher 50 K SNP array to develop a high‐density linkage map with 37 262 markers assigned to 34 linkage groups in 33 autosomes and the Z chromosome. The best‐order map contained 4215 markers, with a total distance of 3132 cM and a mean genetic distance between markers of 0.12 cM . Facilitated by the array being designed to include markers from most scaffolds, we obtained a second‐generation assembly of the flycatcher genome that approaches full chromosome sequences (N50 super‐scaffold size 20.2 Mb and with 1.042 Gb (of 1.116 Gb) anchored to and mostly ordered and oriented along chromosomes). We found that flycatcher and zebra finch chromosomes are entirely syntenic but that inversions at mean rates of 1.5–2.0 event (6.6–7.5 Mb) per My have changed the organization within chromosomes, rates high enough for inversions to potentially have been involved with many speciation events during avian evolution. The mean recombination rate was 3.1 cM /Mb and correlated closely with chromosome size, from 2 cM /Mb for chromosomes >100 Mb to >10 cM /Mb for chromosomes <10 Mb. This size dependence seemed entirely due to an obligate recombination event per chromosome; if 50 cM was subtracted from the genetic lengths of chromosomes, the rate per physical unit DNA was constant across chromosomes. Flycatcher recombination rate showed similar variation along chromosomes as chicken but lacked the large interior recombination deserts characteristic of zebra finch chromosomes.     

Entomopathogenic nematodes fail to parasitize the woolly apple aphid Eriosoma lanigerum as their symbiotic bacteria are suppressed

The restriction of effective insecticides has facilitated the woolly apple aphid (WAA) Eriosoma lanigerum to become a major pest in apple orchards in Western Europe. It has also promoted alternative control strategies such as the use of entomopathogenic nematodes (EPN). We evaluated the control capacity of six commercially available EPN, viz. Heterorhabditis bacteriophora, Heterorhabditis megidis, Steinernema carpocapsae, Steinernema feltiae, Steinernema glaseri and Steinernema kraussei. We assessed the potential of these EPN to colonize and parasitize E. lanigerum in an in vitro multiwell test. Only S. carpocapsae caused higher mortality (20–40%) than the control treatment (water). However, the mortality observed with S. carpocapsae was found to be a test artefact and not induced by its specific entomopathogenic activity. A similar mortality range was recorded when applying the non‐entomopathogenic nematode Pratylenchus thornei in the same multiwell test set‐up. This result warrants careful interpretation of parasitism in these artificial test conditions. The failure of EPN activity was supported in further experiments by frequently finding S. carpocapsae inside living WAA. The presence of the EPN had no effect on aphid reproduction as numbers of ‘large’ embryos in EPN‐colonized and non‐colonized females were similar. In addition, the dauer juveniles did not recover in E. lanigerum reflecting that S. carpocapsae could not develop inside the WAA. We further demonstrated that growth of the EPN‐symbiotic bacteria Xenorhabdus nematophila and Photorhabdus luminescens is inhibited by the body fluid of the WAA, and we speculate that this antibacterial activity is the cause of the unsuccessful parasitization of the WAA by the EPN. This antibiosis inside the body of E. lanigerum would prevent production of the endotoxins by the bacterial symbionts that are essential for entomopathogenicity and insect control.     

Chromosomal‐level genomes of three rice planthoppers provide new insights into sex chromosome evolution

The brown planthopper Nilaparvata lugens, white‐backed planthopper Sogatella furcifera, and small brown planthopper Laodelphax striatellus are three major insect pests of rice. They are genetically close; however, they differ in several ecological traits such as host range, migration capacity, and in their sex chromosomes. Though the draft genome of these three planthoppers have been previously released, the quality of genome assemblies need to be improved. The absence of chromosome‐level genome resources has hindered in‐depth research of these three species. Here, we performed a de novo genome assembly for N. lugens to increase its genome assembly quality with PacBio and Illumina platforms, increasing the contig N50 to 589.46 Kb. Then, with the new N. lugens genome and previously reported S. furcifera and L. striatellus genome assemblies, we generated chromosome‐level scaffold assemblies of these three planthopper species using HiC scaffolding technique. The scaffold N50s significantly increased to 77.63 Mb, 43.36 Mb and 29.24 Mb for N. lugens, S. furcifera and L. striatellus, respectively. To identify sex chromosomes of these three planthopper species, we carried out genome re‐sequencing of males and females and successfully determined the X and Y chromosomes for N. lugens, and X chromosome for S. furcifera and L. striatellus. The gene content of the sex chromosomes showed high diversity among these three planthoppers suggesting the rapid evolution of sex‐linked genes, and all chromosomes showed high synteny. The chromosome‐level genome assemblies of three planthoppers would provide a valuable resource for a broad range of future research in molecular ecology, and subsequently benefits development of modern pest control strategies.     

Chromosome-level genome assembly of an agricultural pest,the rice leaffolder Cnaphalocrocis exigua (Crambidae,Lepidoptera)

The rice leaffolder Cnaphalocrocis exigua (Crambidae, Lepidoptera) is an important agricultural pest that damages rice crops and other members of related grass families. C. exigua exhibits a very similar morphological phenotype and feeding behaviour to C. medinalis, another species of rice leaffolder whose genome was recently reported. However, genomic information for C. exigua remains extremely limited. Here, we used a hybrid strategy combining different sequencing technologies, including Illumina, PacBio, 10× Genomics, and Hi – C scaffolding, to generate a high-quality chromosome-level genome assembly of C. exigua. We initially obtained a 798.8 Mb assembly with a contig N50 size of 2.9 Mb, and the N50 size was subsequently increased to 25.7 Mb using Hi – C technology to anchor 1413 scaffolds to 32 chromosomes. We detected a total of 97.7% Benchmarking Universal Single-Copy Orthologues (BUSCO) in the genome assembly, which was comprised of ~52% repetitive sequence and annotated 14,922 protein-coding genes. Of note, the Z and W sex chromosomes were assembled and identified. A comparative genomic analysis demonstrated that despite the high synteny observed between the two rice leaffolders, the species have distinct genomic features associated with expansion and contraction of gene families and selection pressure. In summary, our chromosome-level genome assembly and comparative genomic analysis of C. exigua provide novel insights into the evolution and ecology of this rice insect pests and offer useful information for pest control.     

A chromosome‐level genome assembly of the parasitoid wasp Pteromalus puparum

Parasitoid wasps represent a large proportion of hymenopteran species. They have complex evolutionary histories and are important biocontrol agents. To advance parasitoid research, a combination of Illumina short‐read, PacBio long‐read and Hi‐C scaffolding technologies was used to develop a high‐quality chromosome‐level genome assembly for Pteromalus puparum, which is an important pupal endoparasitoid of caterpillar pests. The chromosome‐level assembly has aided in studies of venom and detoxification genes. The assembled genome size is 338 Mb with a contig N50 of 38.7 kb and a scaffold N50 of 1.16 Mb. Hi‐C analysis assembled scaffolds onto five chromosomes and raised the scaffold N50 to 65.8 Mb, with more than 96% of assembled bases located on chromosomes. Gene annotation was assisted by RNA sequencing for the two sexes and four different life stages. Analysis detected 98% of the BUSCO (Benchmarking Universal Single‐Copy Orthologs) gene set, supporting a high‐quality assembly and annotation. In total, 40.1% (135.6 Mb) of the assembly is composed of repetitive sequences, and 14,946 protein‐coding genes were identified. Although venom genes play important roles in parasitoid biology, their spatial distribution on chromosomes was poorly understood. Mapping has revealed venom gene tandem arrays for serine proteases, pancreatic lipase‐related proteins and kynurenine–oxoglutarate transaminases, which have amplified in the P. puparum lineage after divergence from its common ancestor with Nasonia vitripennis. In addition, there is a large expansion of P450 genes in P. puparum. These examples illustrate how chromosome‐level genome assembly can provide a valuable resource for molecular, evolutionary and biocontrol studies of parasitoid wasps.     

Assembly and RNA‐free annotation of highly heterozygous genomes: The case of the thick‐billed murre (Uria lomvia)

Thanks to a dramatic reduction in sequencing costs followed by a rapid development of bioinformatics tools, genome assembly and annotation have become accessible to many researchers in recent years. Among tetrapods, birds have genomes that display many features that facilitate their assembly and annotation, such as small genome size, low number of repeats and highly conserved genomic structure. However, we found that high genomic heterozygosity could have a great impact on the quality of the genome assembly of the thick‐billed murre (Uria lomvia), an arctic colonial seabird. In this study, we tested the performance of three genome assemblers, ray /sscape , soapdenovo 2 and platanus , in assembling the highly heterozygous genome of the thick‐billed murre. Our results show that platanus , an assembler specifically designed for heterozygous genomes, outperforms the other two approaches and produces a highly contiguous (N50 = 15.8 Mb) and complete genome assembly (93% presence of genes from the Benchmarking Universal Single Copy Ortholog [BUSCO] gene set). Additionally, we annotated the thick‐billed murre genome using a homology‐based approach that takes advantage of the genomic resources available for birds and other taxa. Our study will be useful for those researchers who are approaching assembly and annotation of highly heterozygous genomes, or genomes of species of conservation concern, and/or who have limited financial resources.     

Finding Nemo’s Genes: A chromosome‐scale reference assembly of the genome of the orange clownfish Amphiprion percula

The iconic orange clownfish, Amphiprion percula, is a model organism for studying the ecology and evolution of reef fishes, including patterns of population connectivity, sex change, social organization, habitat selection and adaptation to climate change. Notably, the orange clownfish is the only reef fish for which a complete larval dispersal kernel has been established and was the first fish species for which it was demonstrated that antipredator responses of reef fishes could be impaired by ocean acidification. Despite its importance, molecular resources for this species remain scarce and until now it lacked a reference genome assembly. Here, we present a de novo chromosome‐scale assembly of the genome of the orange clownfish Amphiprion percula. We utilized single‐molecule real‐time sequencing technology from Pacific Biosciences to produce an initial polished assembly comprised of 1,414 contigs, with a contig N50 length of 1.86 Mb. Using Hi‐C‐based chromatin contact maps, 98% of the genome assembly were placed into 24 chromosomes, resulting in a final assembly of 908.8 Mb in length with contig and scaffold N50s of 3.12 and 38.4 Mb, respectively. This makes it one of the most contiguous and complete fish genome assemblies currently available. The genome was annotated with 26,597 protein‐coding genes and contains 96% of the core set of conserved actinopterygian orthologs. The availability of this reference genome assembly as a community resource will further strengthen the role of the orange clownfish as a model species for research on the ecology and evolution of reef fishes.     

Genome mapping of three major resistance genes to woolly apple aphid (Eriosoma lanigerum Hausm.)

Woolly apple aphid (WAA; Eriosoma lanigerum Hausm.) can be a major economic problem to apple growers in most parts of the world, and resistance breeding provides a sustainable means to control this pest. We report molecular markers for three genes conferring WAA resistance and placing them on two linkage groups (LG) on the genetic map of apple. The Er1 and Er2 genes derived from ‘Northern Spy’ and ‘Robusta 5,’ respectively, are the two major genes that breeders have used to date to improve the resistance of apple rootstocks to this pest. The gene Er3, from ‘Aotea 1’ (an accession classified as Malus sieboldii), is a new major gene for WAA resistance. Genetic markers linked to the Er1 and Er3 genes were identified by screening random amplification of polymorphic deoxyribonucleic acid (DNA; RAPD) markers across DNA bulks from resistant and susceptible plants from populations segregating for these genes. The closest RAPD markers were converted into sequence-characterized amplified region markers and the genome location of these two genes was assigned to LG 08 by aligning the maps around the genes with a reference map of ‘Discovery’ using microsatellite markers. The Er2 gene was located on LG 17 of ‘Robusta 5’ using a genetic map developed in a M.9 × ‘Robusta 5’ progeny. Markers for each of the genes were validated for their usefulness for marker-assisted selection in separate populations. The potential use of the genetic markers for these genes in the breeding of apple cultivars with durable resistance to WAA is discussed.     

A chromosome‐level genome assembly reveals the genetic basis of cold tolerance in a notorious rice insect pest,Chilo suppressalis

The rice stem borer, Chilo suppressalis, is one of the most damaging insect pests to rice production worldwide. Although C. suppressalis has been the focus of numerous studies examining cold tolerance and diapause, plant–insect interactions, pesticide targets and resistance, and the development of RNAi‐mediated pest management, the absence of a high‐quality genome has limited deeper insights. To address this limitation, we generated a draft C. suppressalis genome constructed from both Illumina and PacBio sequences. The assembled genome size was 824.35 Mb with a contig N₅₀ of 307 kb and a scaffold N₅₀ of 1.75 Mb. Hi‐C scaffolding assigned 99.2% of the bases to one of 29 chromosomes. Based on universal single‐copy orthologues (BUSCO), the draft genome assembly was estimated to be 97% complete and is predicted to encompass 15,653 protein‐coding genes. Cold tolerance is an extreme survival strategy found in animals. However, little is known regarding the genetic basis of the winter ecology of C. suppressalis. Here, we focused our orthologous analysis on those gene families associated with animal cold tolerance. Our finding provided the first genomic evidence revealing specific cold‐tolerant strategies in C. suppressalis, including those involved in glucose‐originated glycerol biosynthesis, triacylglycerol‐originated glycerol biosynthesis, fatty acid synthesis and trehalose transport‐intermediate cold tolerance. The high‐quality C. suppressalis genome provides a valuable resource for research into a broad range of areas in molecular ecology, and subsequently benefits developing modern pest control strategies.     

De novo assembly of a chromosome‐level reference genome of red‐spotted grouper (Epinephelus akaara) using nanopore sequencing and Hi‐C

The red‐spotted grouper Epinephelus akaara (E. akaara) is one of the most economically important marine fish in China, Japan and South‐East Asia and is a threatened species. The species is also considered a good model for studies of sex inversion, development, genetic diversity and immunity. Despite its importance, molecular resources for E. akaara remain limited and no reference genome has been published to date. In this study, we constructed a chromosome‐level reference genome of E. akaara by taking advantage of long‐read single‐molecule sequencing and de novo assembly by Oxford Nanopore Technology (ONT) and Hi‐C. A red‐spotted grouper genome of 1.135 Gb was assembled from a total of 106.29 Gb polished Nanopore sequence (GridION, ONT), equivalent to 96‐fold genome coverage. The assembled genome represents 96.8% completeness (BUSCO) with a contig N50 length of 5.25 Mb and a longest contig of 25.75 Mb. The contigs were clustered and ordered onto 24 pseudochromosomes covering approximately 95.55% of the genome assembly with Hi‐C data, with a scaffold N50 length of 46.03 Mb. The genome contained 43.02% repeat sequences and 5,480 noncoding RNAs. Furthermore, combined with several RNA‐seq data sets, 23,808 (99.5%) genes were functionally annotated from a total of 23,923 predicted protein‐coding sequences. The high‐quality chromosome‐level reference genome of E. akaara was assembled for the first time and will be a valuable resource for molecular breeding and functional genomics studies of red‐spotted grouper in the future.     

A chromosome‐level genome assembly of the giant grouper (Epinephelus lanceolatus) provides insights into its innate immunity and rapid growth

The giant grouper (Epinephelus lanceolatus) is the largest coral reef teleost, with a native range that spans temperate and tropical waters in the Pacific and the Indian Oceans. It is cultured artificially and used as a breeding species in aquaculture due to its rapid growth rate. Here we report a giant grouper genome assembled at the chromosome scale from sequences generated using Illumina and high‐throughput chromatin conformation capture (Hi‐C) technology. The assembly comprised 1.086 Gb, with 98.4% of the scaffold sequences anchored into 24 chromosomes. The contig and scaffold N50 values were 119.9 kb and 46.2 Mb, respectively. The assembly is of high integrity, including 96.4% universal single‐copy orthologues based on BUSCO analysis. Through chromosome‐scale evolution analysis, we identified alignments of six giant grouper chromosomes to three stickleback chromosomes and some of the genes located within the breakpoints of reshuffling events may related to development and growth. From the 24,718 protein‐coding genes, we found that several gene families related to innate immunity and glycan biosynthesis were significantly expanded in the giant grouper genome compared to other teleost genomes. In addition, we identified several genes related to the hormone signalling pathway and innate immunity that have experienced positive selection or accelerated evolution, implicating their roles in immune defence and fast growth of the species. The high‐quality genome assembly will provide a valuable genomic resource for further biological and evolutionary studies, and useful genomic tools for breeding of the giant grouper.