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.     

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.     

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.     

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.     

Chromosome‐level genome assembly of the razor clam Sinonovacula constricta (Lamarck, 1818)

Bivalves, a highly diverse and the most evolutionarily successful class of invertebrates native to aquatic habitats, provide valuable molecular resources for understanding the evolutionary adaptation and aquatic ecology. Here, we reported a high‐quality chromosome‐level genome assembly of the razor clam Sinonovacula constricta using Pacific Bioscience single‐molecule real‐time sequencing, Illumina paired‐end sequencing, 10X Genomics linked‐reads and Hi‐C reads. The genome size was 1,220.85 Mb, containing scaffold N50 of 65.93 Mb and contig N50 of 976.94 Kb. A total of 899 complete (91.92%) and seven partial (0.72%) matches of the 978 metazoa Benchmarking Universal Single‐Copy Orthologs were determined in this genome assembly. And Hi‐C scaffolding of the genome resulted in 19 pseudochromosomes. A total of 28,594 protein‐coding genes were predicted in the S. constricta genome, of which 25,413 genes (88.88%) were functionally annotated. In addition, 39.79% of the assembled genome was composed of repetitive sequences, and 4,372 noncoding RNAs were identified. The enrichment analyses of the significantly expanded and contracted genes suggested an evolutionary adaptation of S. constricta to highly stressful living environments. In summary, the genomic resources generated in this work not only provide a valuable reference genome for investigating the molecular mechanisms of S. constricta biological functions and evolutionary adaptation, but also facilitate its genetic improvement and disease treatment. Meanwhile, the obtained genome greatly improves our understanding of the genetics of molluscs and their comparative evolution.     

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.     

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.     

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.     

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 sequence of M6, a diploid inbred clone of the high‐glycoalkaloid‐producing tuber‐bearing potato species Solanum chacoense,reveals residual heterozygosity

Cultivated potato (Solanum tuberosum L.) is a highly heterozygous autotetraploid that presents challenges in genome analyses and breeding. Wild potato species serve as a resource for the introgression of important agronomic traits into cultivated potato. One key species is Solanum chacoense and the diploid, inbred clone M6, which is self‐compatible and has desirable tuber market quality and disease resistance traits. Sequencing and assembly of the genome of the M6 clone of S. chacoense generated an assembly of 825 767 562 bp in 8260 scaffolds with an N50 scaffold size of 713 602 bp. Pseudomolecule construction anchored 508 Mb of the genome assembly into 12 chromosomes. Genome annotation yielded 49 124 high‐confidence gene models representing 37 740 genes. Comparative analyses of the M6 genome with six other Solanaceae species revealed a core set of 158 367 Solanaceae genes and 1897 genes unique to three potato species. Analysis of single nucleotide polymorphisms across the M6 genome revealed enhanced residual heterozygosity on chromosomes 4, 8 and 9 relative to the other chromosomes. Access to the M6 genome provides a resource for identification of key genes for important agronomic traits and aids in genome‐enabled development of inbred diploid potatoes with the potential to accelerate potato breeding.     

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.     

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.     

Chromosome‐level de novo genome assembly of Sarcophaga peregrina provides insights into the evolutionary adaptation of flesh flies

Sarcophaga peregrina is considered to be of great ecological, medical and forensic significance, and has unusual biological characteristics such as an ovoviviparous reproductive pattern and adaptation to feed on carrion. The availability of a high‐quality genome will help to further reveal the mechanisms underlying these charcateristics. Here we present a de novo‐assembled genome at chromosome scale for S. peregrina. The final assembled genome was 560.31 Mb with contig N50 of 3.84 Mb. Hi‐C scaffolding reliably anchored six pseudochromosomes, accounting for 97.76% of the assembled genome. Moreover, 45.70% of repeat elements were identified in the genome. A total of 14,476 protein‐coding genes were functionally annotated, accounting for 92.14% of all predicted genes. Phylogenetic analysis indicated that S. peregrina and S. bullata diverged ~ 7.14 million years ago. Comparative genomic analysis revealed expanded and positively selected genes related to biological features that aid in clarifying its ovoviviparous reproduction and carrion‐feeding adaptations, such as lipid metabolism, olfactory receptor activity, antioxidant enzymes, proteolysis and serine‐type endopeptidase activity. Protein‐coding genes associated with ovoviparity, such as yolk proteins, transferrin and acid sphingomyelinase, were identified. This study provides a valuable genomic resource for S. peregrina, and sheds insight into further revealing the underlying molecular mechanisms of adaptive evolution.     

De novo assembly of a wild pear (Pyrus betuleafolia) genome

China is the origin and evolutionary centre of Oriental pears. Pyrus betuleafolia is a wild species native to China and distributed in the northern region, and it is widely used as rootstock. Here, we report the de novo assembly of the genome of P. betuleafolia‐Shanxi Duli using an integrated strategy that combines PacBio sequencing, BioNano mapping and chromosome conformation capture (Hi‐C) sequencing. The genome assembly size was 532.7 Mb, with a contig N50 of 1.57 Mb. A total of 59 552 protein‐coding genes and 247.4 Mb of repetitive sequences were annotated for this genome. The expansion genes in P. betuleafolia were significantly enriched in secondary metabolism, which may account for the organism's considerable environmental adaptability. An alignment analysis of orthologous genes showed that fruit size, sugar metabolism and transport, and photosynthetic efficiency were positively selected in Oriental pear during domestication. A total of 573 nucleotide‐binding site (NBS)‐type resistance gene analogues (RGAs) were identified in the P. betuleafolia genome, 150 of which are TIR‐NBS‐LRR (TNL)‐type genes, which represented the greatest number of TNL‐type genes among the published Rosaceae genomes and explained the strong disease resistance of this wild species. The study of flavour metabolism‐related genes showed that the anthocyanidin reductase (ANR) metabolic pathway affected the astringency of pear fruit and that sorbitol transporter (SOT) transmembrane transport may be the main factor affecting the accumulation of soluble organic matter. This high‐quality P. betuleafolia genome provides a valuable resource for the utilization of wild pear in fundamental pear studies and breeding.     

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 reference genome of X12, a highly virulent race of the soybean cyst nematode Heterodera glycines

Soybean cyst nematode (SCN, Heterodera glycines) is a major pest of soybean that is spreading across major soybean production regions worldwide. Increased SCN virulence has recently been observed in both the United States and China. However, no study has reported a genome assembly for H. glycines at the chromosome scale. Herein, the first chromosome‐level reference genome of X12, an unusual SCN race with high infection ability, is presented. Using whole‐genome shotgun (WGS) sequencing, Pacific Biosciences (PacBio) sequencing, Illumina paired‐end sequencing, 10X Genomics linked reads and high‐throughput chromatin conformation capture (Hi‐C) genome scaffolding techniques, a 141.01‐megabase (Mb) assembled genome was obtained with scaffold and contig N50 sizes of 16.27 Mb and 330.54 kilobases (kb), respectively. The assembly showed high integrity and quality, with over 90% of Illumina reads mapped to the genome. The assembly quality was evaluated using Core Eukaryotic Genes Mapping Approach and Benchmarking Universal Single‐Copy Orthologs. A total of 11,882 genes were predicted using de novo, homolog and RNAseq data generated from eggs, second‐stage juveniles (J2), third‐stage juveniles (J3) and fourth‐stage juveniles (J4) of X12, and 79.0% of homologous sequences were annotated in the genome. These high‐quality X12 genome data will provide valuable resources for research in a broad range of areas, including fundamental nematode biology, SCN–plant interactions and co‐evolution, and also contribute to the development of technology for overall SCN management.     

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.     

Chromosome‐level reference genome assembly and gene editing of the dead‐leaf butterfly Kallima inachus

The leaf resemblance of Kallima (Nymphalidae) butterflies is an important ecological adaptive mechanism that increases their survival. However, the genetic mechanism underlying ecological adaptation remains unclear owing to a dearth of genomic information. Here, we determined the karyotype (n = 31) of the dead‐leaf butterfly Kallima inachus, and generated a high‐quality, chromosome‐level assembly (568.92 Mb; contig N50: 19.20 Mb). We also identified candidate Z and W chromosomes. To our knowledge, this is the first study to report on these aspects of this species. In the assembled genome, 15,309 protein‐coding genes and 49.86% repeat elements were annotated. Phylogenetic analysis showed that K. inachus diverged from Melitaea cinxia (no leaf resemblance), both of which are in Nymphalinae, around 40 million years ago. Demographic analysis indicated that the effective population size of K. inachus decreased during the last interglacial period in the Pleistocene. The wings of adults with the pigmentary gene ebony knocked out using CRISPR/Cas9 showed phenotypes in which the orange dorsal region and entire ventral surface darkened, suggesting its vital role in the ecological adaption of dead‐leaf butterflies. Our results provide important genome resources for investigating the genetic mechanism underlying protective resemblance in dead‐leaf butterflies and insights into the molecular basis of protective coloration.