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.     

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 greenfin horse‐faced filefish (Thamnaconus septentrionalis) using Oxford Nanopore PromethION sequencing and Hi‐C technology

The greenfin horse‐faced filefish, Thamnaconus septentrionalis, is a valuable commercial fish species that is widely distributed in the Indo‐West Pacific Ocean. This fish has characteristic blue–green fins, rough skin and a spine‐like first dorsal fin. Thamnaconus septentrionalis is of conservation concern because its population has declined sharply, and it is an important marine aquaculture fish species in China. Genomic resources for the filefish are lacking, and no reference genome has been released. In this study, the first chromosome‐level genome of T. septentrionalis was constructed using nanopore sequencing and Hi‐C technology. A total of 50.95 Gb polished nanopore sequences were generated and were assembled into a 474.31‐Mb genome, accounting for 96.45% of the estimated genome size of this filefish. The assembled genome contained only 242 contigs, and the achieved contig N50 was 22.46 Mb, a surprisingly high value among all sequenced fish species. Hi‐C scaffolding of the genome resulted in 20 pseudochromosomes containing 99.44% of the total assembled sequences. The genome contained 67.35 Mb of repeat sequences, accounting for 14.2% of the assembly. A total of 22,067 protein‐coding genes were predicted, 94.82% of which were successfully annotated with putative functions. Furthermore, a phylogenetic tree was constructed using 1,872 single‐copy orthologous genes, and 67 unique gene families were identified in the filefish genome. This high‐quality assembled genome will be a valuable resource for a range of future genomic, conservation and breeding studies of T. septentrionalis.     

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.     

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.     

Chromosome‐level genome assembly of an important pine defoliator,Dendrolimus punctatus (Lepidoptera; Lasiocampidae)

Dendrolimus spp. are important destructive pests of conifer forests, and Dendrolimus punctatus Walker (Lepidoptera; Lasiocampidae) is the most widely distributed Dendrolimus species. During periodic outbreaks, this species is said to make “fire without smoke” because large areas of pine forest can be quickly and heavily damaged. Yet, little is known about the molecular mechanisms that underlie the unique ecological characteristics of this forest insect. Here, we combined Pacific Biosciences (PacBio) RSII single‐molecule long reads and high‐throughput chromosome conformation capture (Hi‐C) genomics‐linked reads to produce a high‐quality, chromosome‐level reference genome for D. punctatus. The final assembly was 614 Mb with contig and scaffold N50 values of 1.39 and 22.15 Mb, respectively, and 96.96% of the contigs anchored onto 30 chromosomes. Based on the prediction, this genome contained 17,593 protein‐coding genes and 56.16% repetitive sequences. Phylogenetic analyses indicated that D. punctatus diverged from the common ancestor of Hyphantria cunea, Spodoptera litura and Thaumetopoea pityocampa ~ 108.91 million years ago. Many gene families that were expanded in the D. punctatus genome were significantly enriched for the xenobiotic biodegradation system, especially the cytochrome P450 gene family. This high‐quality, chromosome‐level reference genome will be a valuable resource for understanding mechanisms of D. punctatus outbreak and host resistance adaption. Because this is the first Lasiocampidae insect genome to be sequenced, it also will serve as a reference for further comparative genomics.     

A chromosome‐level genome assembly of the woolly apple aphid,Eriosoma lanigerum Hausmann (Hemiptera: Aphididae)

Woolly apple aphid (WAA, Eriosoma lanigerum Hausmann) (Hemiptera: Aphididae) is a major pest of apple trees (Malus domestica, order Rosales) and is critical to the economics of the apple industry in most parts of the world. Here, we generated a chromosome‐level genome assembly of WAA—representing the first genome sequence from the aphid subfamily Eriosomatinae—using a combination of 10X Genomics linked‐reads and in vivo Hi‐C data. The final genome assembly is 327 Mb, with 91% of the assembled sequences anchored into six chromosomes. The contig and scaffold N50 values are 158 kb and 71 Mb, respectively, and we predicted a total of 28,186 protein‐coding genes. The assembly is highly complete, including 97% of conserved arthropod single‐copy orthologues based on Benchmarking Universal Single‐Copy Orthologs (busco ) analysis. Phylogenomic analysis of WAA and nine previously published aphid genomes, spanning four aphid tribes and three subfamilies, reveals that the tribe Eriosomatini (represented by WAA) is recovered as a sister group to Aphidini + Macrosiphini (subfamily Aphidinae). We identified syntenic blocks of genes between our WAA assembly and the genomes of other aphid species and find that two WAA chromosomes (El5 and El6) map to the conserved Macrosiphini and Aphidini X chromosome. Our high‐quality WAA genome assembly and annotation provides a valuable resource for research in a broad range of areas such as comparative and population genomics, insect–plant interactions and pest resistance management.     

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 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.     

Apolygus lucorum genome provides insights into omnivorousness and mesophyll feeding

Apolygus lucorum (Miridae) is an omnivorous pest that occurs worldwide and is notorious for the serious damage it causes to various crops and substantial economic losses. Although some studies have examined the biological characteristics of the mirid bug, no reference genome is available in Miridae, limiting in‐depth studies of this pest. Here, we present a chromosome‐scale reference genome of A. lucorum, the first sequenced Miridae species. The assembled genome size was 1.02 Gb with a contig N50 of 785 kb. With Hi‐C scaffolding, 1,016 Mb contig sequences were clustered, ordered and assembled into 17 large scaffolds with scaffold N50 length 68 Mb, each corresponding to a natural chromosome. Numerous transposable elements occur in this genome and contribute to the large genome size. Expansions of genes associated with omnivorousness and mesophyll feeding such as those related to digestion, chemosensory perception, and detoxification were observed in A. lucorum, suggesting that gene expansion contributed to its strong environmental adaptability and severe harm to crops. We clarified that a salivary enzyme polygalacturonase is unique in mirid bugs and has significantly expanded in A. lucorum, which may contribute to leaf damage from this pest. The reference genome of A. lucorum not only facilitates biological studies of Hemiptera as well as an understanding of the damage mechanism of mesophyll feeding, but also provides a basis on which to develop efficient control technologies for mirid bugs.     

Chromosome‐scale pseudomolecules refined by optical,physical and genetic maps in flax

Genomes of varying sizes have been sequenced with next‐generation sequencing platforms. However, most reference sequences include draft unordered scaffolds containing chimeras caused by mis‐scaffolding. A BioNano genome (BNG) optical map was constructed to improve the previously sequenced flax genome (Linum usitatissimum L., 2n = 30, about 373 Mb), which consisted of 3852 scaffolds larger than 1 kb and totalling 300.6 Mb. The high‐resolution BNG map of cv. CDC Bethune totalled 317 Mb and consisted of 251 BNG contigs with an N50 of 2.15 Mb. A total of 622 scaffolds (286.6 Mb, 94.9%) aligned to 211 BNG contigs (298.6 Mb, 94.2%). Of those, 99 scaffolds, diagnosed to contain assembly errors, were refined into 225 new scaffolds. Using the newly refined scaffold sequences and the validated bacterial artificial chromosome‐based physical map of CDC Bethune, the 211 BNG contigs were scaffolded into 94 super‐BNG contigs (N50 of 6.64 Mb) that were further assigned to the 15 flax chromosomes using the genetic map. The pseudomolecules total about 316 Mb, with individual chromosomes of 15.6 to 29.4 Mb, and cover 97% of the annotated genes. Evidence from the chromosome‐scale pseudomolecules suggests that flax has undergone palaeopolyploidization and mesopolyploidization events, followed by rearrangements and deletions or fusion of chromosome arms from an ancient progenitor with a haploid chromosome number of eight.     

Chromosome‐level genome assembly of Triplophysa tibetana,a fish adapted to the harsh high‐altitude environment of the Tibetan Plateau

Triplophysa is an endemic fish genus of the Tibetan Plateau in China. Triplophysa tibetana, which lives at a recorded altitude of ~4,000 m and plays an important role in the highland aquatic ecosystem, serves as an excellent model for investigating high‐altitude environmental adaptation. However, evolutionary and conservation studies of T. tibetana have been limited by scarce genomic resources for the genus Triplophysa. In the present study, we applied PacBio sequencing and the Hi‐C technique to assemble the T. tibetana genome. A 652‐Mb genome with 1,325 contigs with an N50 length of 3.1 Mb was obtained. The 1,137 contigs were further assembled into 25 chromosomes, representing 98.7% and 80.47% of all contigs at the base and sequence number level, respectively. Approximately 260 Mb of sequence, accounting for ~39.8% of the genome, was identified as repetitive elements. DNA transposons (16.3%), long interspersed nuclear elements (12.4%) and long terminal repeats (11.0%) were the most repetitive types. In total, 24,372 protein‐coding genes were predicted in the genome, and ~95% of the genes were functionally annotated via a search in public databases. Using whole genome sequence information, we found that T. tibetana diverged from its common ancestor with Danio rerio ~121.4 million years ago. The high‐quality genome assembled in this work not only provides a valuable genomic resource for future population and conservation studies of T. tibetana, but it also lays a solid foundation for further investigation into the mechanisms of environmental adaptation of endemic fishes in the Tibetan Plateau.     

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.     

Chromosome‐level genome assembly of the predator Propylea japonica to understand its tolerance to insecticides and high temperatures

The ladybird beetle Propylea japonica is an important natural enemy in agro‐ecological systems. Studies on the strong tolerance of P. japonica to high temperatures and insecticides, and its population and phenotype diversity have recently increased. However, abundant genome resources for obtaining insights into stress‐resistance mechanisms and genetic intra‐species diversity for P. japonica are lacking. Here, we constructed the P. japonica genome maps using Pacific Bioscience (PacBio) and Illumina sequencing technologies. The genome size was 850.90 Mb with a contig N50 of 813.13 kb. The Hi‐C sequence data were used to upgrade draft genome assemblies; 4,777 contigs were assembled to 10 chromosomes; and the final draft genome assembly was 803.93 Mb with a contig N50 of 813.98 kb and a scaffold N50 of 100.34 Mb. Approximately 495.38 Mb of repeated sequences was annotated. The 18,018 protein‐coding genes were predicted, of which 95.78% were functionally annotated, and 1,407 genes were species‐specific. The phylogenetic analysis showed that P. japonica diverged from the ancestor of Anoplophora glabripennis and Tribolium castaneum ~ 236.21 million years ago. We detected that some important gene families involved in detoxification of pesticides and tolerance to heat stress were expanded in P. japonica, especially cytochrome P450 and Hsp70 genes. Overall, the high‐quality draft genome sequence of P. japonica will provide invaluable resource for understanding the molecular mechanisms of stress resistance and will facilitate the research on population genetics, evolution and phylogeny of Coccinellidae. This genome will also provide new avenues for conserving the diversity of predator insects.     

Improving Illumina assemblies with Hi‐C and long reads: An example with the North African dromedary

Researchers have assembled thousands of eukaryotic genomes using Illumina reads, but traditional mate‐pair libraries cannot span all repetitive elements, resulting in highly fragmented assemblies. However, both chromosome conformation capture techniques, such as Hi‐C and Dovetail Genomics Chicago libraries and long‐read sequencing, such as Pacific Biosciences and Oxford Nanopore, help span and resolve repetitive regions and therefore improve genome assemblies. One important livestock species of arid regions that does not have a high‐quality contiguous reference genome is the dromedary (Camelus dromedarius). Draft genomes exist but are highly fragmented, and a high‐quality reference genome is needed to understand adaptation to desert environments and artificial selection during domestication. Dromedaries are among the last livestock species to have been domesticated, and together with wild and domestic Bactrian camels, they are the only representatives of the Camelini tribe, which highlights their evolutionary significance. Here we describe our efforts to improve the North African dromedary genome. We used Chicago and Hi‐C sequencing libraries from Dovetail Genomics to resolve the order of previously assembled contigs, producing almost chromosome‐level scaffolds. Remaining gaps were filled with Pacific Biosciences long reads, and then scaffolds were comparatively mapped to chromosomes. Long reads added 99.32 Mbp to the total length of the new assembly. Dovetail Chicago and Hi‐C libraries increased the longest scaffold over 12‐fold, from 9.71 Mbp to 124.99 Mbp and the scaffold N50 over 50‐fold, from 1.48 Mbp to 75.02 Mbp. We demonstrate that Illumina de novo assemblies can be substantially upgraded by combining chromosome conformation capture and long‐read sequencing.