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. 2025 Jan 14;10:12. [Version 1] doi: 10.12688/wellcomeopenres.23504.1

The genome sequence of the European Corn Borer, Ostrinia nubilalis Hübner, 1796

Douglas Boyes 1, David C Lees 2, Brad S Coates 3; University of Oxford and Wytham Woods Genome Acquisition Lab; Natural History Museum Genome Acquisition Lab; Darwin Tree of Life Barcoding collective; Wellcome Sanger Institute Tree of Life Management, Samples and Laboratory team; Wellcome Sanger Institute Scientific Operations: Sequencing Operations; Wellcome Sanger Institute Tree of Life Core Informatics team; Tree of Life Core Informatics collective; Darwin Tree of Life Consortiuma
PMCID: PMC11851004  PMID: 40007688

Abstract

We present a genome assembly from an individual female specimen of Ostrinia nubilalis (European Corn Borer; Arthropoda; Insecta; Lepidoptera; Crambidae). The genome sequence has a total length of 495.50 megabases. Most of the assembly (99.87%) is scaffolded into 32 chromosomal pseudomolecules, including the Z and W sex chromosomes. The mitochondrial genome has also been assembled and is 15.24 kilobases in length. Gene annotation of this assembly on Ensembl identified 16,780 protein-coding genes.

Keywords: Ostrinia nubilalis, European Corn Borer, genome sequence, chromosomal, Lepidoptera

Species taxonomy

Eukaryota; Opisthokonta; Metazoa; Eumetazoa; Bilateria; Protostomia; Ecdysozoa; Panarthropoda; Arthropoda; Mandibulata; Pancrustacea; Hexapoda; Insecta; Dicondylia; Pterygota; Neoptera; Endopterygota; Amphiesmenoptera; Lepidoptera; Glossata; Neolepidoptera; Heteroneura; Ditrysia; Obtectomera; Pyraloidea; Crambidae; Pyraustinae; Ostrinia; Ostrinia nubilalis Hübner, 1796 (NCBI:txid29057)

Background

The European corn borer (ECB), Ostrinia nubilalis, is a small snout-nosed moth species in the family Crambidae, with an endemic range across Europe, western Asia and Mediterranean region of Africa. Introduction into North America during the early 1900s led to its eventual establishment across areas east of the Rocky Mountains of the United States, and maritime and southern tiers of central plain provinces in Canada. Larval O. nubilalis are reported to feed on over 240 host plants, including cultivated maize, a host plant that suffers significant yield losses ( Hodgson, 1928). Economic costs to farmers in North America were severe, exceeding 1 billion USD in control measures and yield losses ( Mason, 1996). After the commercialisation of transgenic maize hybrids with insecticidal Bacillus thuringiensis ( Bt) insecticidal crystalline (Cry) proteins in 1995, area-wide effects caused widespread population suppression and vastly reduced incidence of associated crop damage ( Hutchison et al., 2010). After nearly 25 years of successful suppression, damage was reported in Canada on transgenic maize expressing Bt Cry1F ( Smith et al., 2019) and Cry1Ab proteins ( Smith & Farhan, 2023).

Like its phylogenetic sister and sibling species, O. furnacalis and O. scapulalis, respectively, male and female O. nubilalis are sexually dimorphic, with males having smaller body size and darker colouration compared to females ( Mutuura & Munroe, 1970). As in other species of Lepidoptera, Ostrinia females are heterogametic (ZW) and males are homogametic (ZZ). Ecological variation exists within and among O. nubilalis populations based on differences in sex pheromone produced by females and responded to by cognate males, and response to environmental cues that determine the number of reproductive generations per year ( Coates et al., 2018). Specifically, E-(NY strain) and Z-race (IA strain) females produce stereoisomers E- and Z-tetradecyl acetate ( E-11Ac and Z-11Ac), respectively ( Klun & Maini, 1979), which is controlled by allelic variation in the autosomal gene, pheromone gland fatty acyl-reductase ( pgfar; Lassance et al., 2013). E- and Z-races are sympatric across the northern Atlantic coast of North America, whereas the geographic range of Z-race also extends westward across major maize growing regions of the United States ( Lambden & Johnson, 2013). Corresponding preferential male attraction is determined by variation in neuronal development that is under direction of E- and Z-race specific differences in the sex-linked transcription factor, bric à brac ( bab) ( Unbehend et al., 2021). The number of mating generations (voltinism) varies across latitudinal clines, with genetic determination of obligatory diapause among fixed single generation univoltine populations in the northern regions. Phenotypic plasticity in a facultative diapause trait exists among multivoltine individuals, resulting in up to five mating generations in the southern regions ( Showers, 1993). These voltine differences are controlled by epistatic interactions between allelic variants of the sex-linked genes period ( per) and pigment dispersing factor receptor ( pdfr), that are part of circadian and circannual rhythm pathways, respectively ( Kozak et al., 2019). Additionally, an approximate 50 Mbp region of suppressed recombination, likely an inversion, occurs on the Z-chromosome of O. nubilalis that includes per, with bab and pdfr located in Z-chromosome regions flanking predicted breakpoints ( Coates et al., 2018; Kozak et al., 2017). Interestingly, this likely inverted region has an elevated frequency within sympatric populations that differ in one or more voltine and/or pheromone traits, suggesting a role in reinforcing assortative mating and persistence of ecotypes.

A chromosome-level genome assembly was generated for O. nubilalis as part of the Darwin Tree of Life project. This assembly will contribute toward the growing resources for studying evolution across Lepidoptera, as well as ecological adaptations and underlying genomic differentiation in the genus Ostrinia.

Genome sequence report

The genome of Ostrinia nubilalis ( Figure 1) was sequenced using Pacific Biosciences single-molecule HiFi long reads, generating a total of 21.96 Gb (gigabases) from 1.50 million reads, providing an estimated 44-fold coverage. Primary assembly contigs were scaffolded with chromosome conformation Hi-C data, which produced 106.66 Gb from 706.37 million reads. Specimen and sequencing details are summarised in Table 1.

Figure 1. Photograph of the Ostrinia nubilalis (ilOstNubi1) specimen used for genome sequencing.

Figure 1.

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Table 1. Specimen and sequencing data for Ostrinia nubilalis.

Project information
Study title Ostrinia nubilalis (European corn borer)
Umbrella BioProject PRJEB65201
Species Ostrinia nubilalis
BioSample SAMEA7701321
NCBI taxonomy ID 29057
Specimen information
Technology ToLID BioSample
accession 		Organism part
PacBio long read
sequencing 		ilOstNubi1 SAMEA7701417 Whole organism
Hi-C sequencing ilOstNubi4 SAMEA112975556 Whole organism
Sequencing information
Platform Run accession Read count Base count (Gb)
Hi-C Illumina NovaSeq
6000 		ERR11872563 7.06e+08 106.66
PacBio Sequel IIe ERR11867203 1.50e+06 21.96
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Assembly errors were corrected by manual curation, including 12 missing joins or mis-joins and four haplotypic duplications. This increased the scaffold number by 1.92%. The final assembly has a total length of 495.50 Mb in 52 sequence scaffolds, with 17 gaps, and a scaffold N50 of 16.5 Mb ( Table 2).

Table 2. Genome assembly data for Ostrinia nubilalis, ilOstNubi1.1.

Genome assembly
Assembly name ilOstNubi1.1
Assembly accession GCA_963855985.1
Accession of alternate haplotype GCA_963856005.1
Span (Mb) 495.50
Number of contigs 70
Number of scaffolds 52
Longest scaffold (Mb) 27.76
Assembly metrics * Benchmark
Contig N50 length (Mb) 15.5 ≥ 1 Mb
Scaffold N50 length (Mb) 16.5 = chromosome N50
Consensus quality (QV) 68.8 (68.4 for combined
haplotypes)		 ≥ 40
k-mer completeness Primary haplotype: 66.52%,
alternate haplotype 61.67%,
both 98.91%
≥ 95%
BUSCO v5.4.3 lineage:
lepidoptera_odb10		 C:98.5%[S:98.2%,D:0.3%],
F:0.5%,M:1.0%,n:5,286		 S > 90%, D < 5%
Percentage of assembly mapped
to chromosomes		 99.87% ≥ 90%
Sex chromosomes ZW localised homologous
pairs
Organelles Mitochondrial genome: 15.24 kb complete single alleles
Genome annotation of assembly GCA_963855985.1 at Ensembl
Number of protein-coding genes 16,780
Number of non-coding genes 10,413
Number of gene transcripts 45,650
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* Assembly metric benchmarks are adapted from Rhie et al. (2021) and the Earth BioGenome Project Report on Assembly Standards September 2024.

** BUSCO scores based on the lepidoptera_odb10 BUSCO set using version 5.4.3. C = complete [S = single copy, D = duplicated], F = fragmented, M = missing, n = number of orthologues in comparison. A full set of BUSCO scores is available at https://blobtoolkit.genomehubs.org/view/Ostrinia_nubilalis/dataset/GCA_963855985.1/busco.

The snail plot in Figure 2 provides a summary of the assembly statistics, indicating the distribution of scaffold lengths and other assembly metrics. Figure 3 shows the distribution of scaffolds by GC proportion and coverage. Figure 4 presents a cumulative assembly plot, with separate curves representing different scaffold subsets assigned to various phyla, illustrating the completeness of the assembly.

Figure 2. Genome assembly of Ostrinia nubilalis, ilOstNubi1.1: metrics.

Figure 2.

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The BlobToolKit snail plot provides an overview of assembly metrics and BUSCO gene completeness. The circumference represents the length of the whole genome sequence, and the main plot is divided into 1,000 bins around the circumference. The outermost blue tracks display the distribution of GC, AT, and N percentages across the bins. Scaffolds are arranged clockwise from longest to shortest and are depicted in dark grey. The longest scaffold is indicated by the red arc, and the deeper orange and pale orange arcs represent the N50 and N90 lengths. A light grey spiral at the centre shows the cumulative scaffold count on a logarithmic scale. A summary of complete, fragmented, duplicated, and missing BUSCO genes in the lepidoptera_odb10 set is presented at the top right. An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/GCA_963855985.1/dataset/GCA_963855985.1/snail.

Figure 3. Genome assembly of Ostrinia nubilalis, ilOstNubi1.1: BlobToolKit GC-coverage plot showing sequence coverage (vertical axis) and GC content (horizontal axis).

Figure 3.

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The circles represent scaffolds, with the size proportional to scaffold length and the colour representing phylum membership. The histograms along the axes display the total length of sequences distributed across different levels of coverage and GC content. An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/GCA_963855985.1/dataset/GCA_963855985.1/blob.

Figure 4. Genome assembly of Ostrinia nubilalis ilOstNubi1.1: BlobToolKit cumulative sequence plot.

Figure 4.

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The grey line shows cumulative length for all scaffolds. Coloured lines show cumulative lengths of scaffolds assigned to each phylum using the buscogenes taxrule. An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/GCA_963855985.1/dataset/GCA_963855985.1/cumulative.

Most of the assembly sequence (99.87%) was assigned to 32 chromosomal-level scaffolds, representing 30 autosomes and the Z and W sex chromosomes. These chromosome-level scaffolds, confirmed by the Hi-C data, are named in order of size ( Figure 5; Table 3). During manual curation it was noted that The Hi-C data was from a male sample, so the exact order of the W chromosome contigs is unknown. The sex chromosomes were identified by coverage.

Figure 5. Genome assembly of Ostrinia nubilalis ilOstNubi1.1: Hi-C contact map of the ilOstNubi1.1 assembly, visualised using HiGlass.

Figure 5.

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Chromosomes are shown in order of size from left to right and top to bottom. An interactive version of this figure may be viewed at https://genome-note-higlass.tol.sanger.ac.uk/l/?d=DXu8jeAmRwO2saQMFKY0Gg.

Table 3. Chromosomal pseudomolecules in the genome assembly of Ostrinia nubilalis, ilOstNubi1.

INSDC
accession		 Name Length
(Mb)		 GC%
OY979734.1 1 19.71 37.5
OY979736.1 2 18.86 37.5
OY979737.1 3 18.82 37.5
OY979738.1 4 18.81 37.0
OY979739.1 5 18.55 37.5
OY979740.1 6 18.36 37.5
OY979741.1 7 17.84 37.5
OY979742.1 8 17.8 37.0
OY979743.1 9 17.37 37.5
OY979744.1 10 16.89 37.5
OY979745.1 11 16.74 37.0
OY979746.1 12 16.57 37.0
OY979747.1 13 16.46 37.5
OY979748.1 14 16.09 37.0
OY979749.1 15 16.03 37.5
OY979750.1 16 15.55 37.5
OY979751.1 17 15.27 37.5
OY979752.1 18 15.24 38.0
OY979753.1 19 14.73 38.0
OY979754.1 20 14.23 37.5
OY979755.1 21 13.82 38.0
OY979756.1 22 13.44 38.0
OY979757.1 23 12.75 37.5
OY979758.1 24 12.57 38.0
OY979759.1 25 11.41 38.0
OY979760.1 26 11.24 38.0
OY979761.1 27 8.94 39.0
OY979762.1 28 8.05 39.0
OY979763.1 29 7.82 39.5
OY979764.1 30 7.5 40.0
OY979735.1 W 12.36 38.0
OY979733.1 Z 27.76 37.0
OY979765.1 MT 0.02 19.0
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While not fully phased, the assembly deposited is of one haplotype. Contigs corresponding to the second haplotype have also been deposited. The mitochondrial genome was also assembled and can be found as a contig within the multifasta file of the genome submission, and as a separate fasta file with accession OY979765.1.

The final assembly has a Quality Value (QV) of 68.8. The k-mer completeness of the primary assembly was found to be 66.52%, and of the alternate haplotype 61.67%, while the k-mer completeness for combined assemblies was 98.91%. BUSCO (v5.4.3) analysis using the lepidoptera_odb10 reference set ( n = 5,286) indicated a completeness score of 98.5% (single = 98.2%, duplicated = 0.3%). The assembly achieves the EBP reference standard of 6.7.68. Other quality metrics are given in Table 2.

Genome annotation report

The Ostrinia nubilalis genome assembly (GCA_963855985.1) was annotated at the European Bioinformatics Institute (EBI) on Ensembl Rapid Release. The resulting annotation includes 45,650 transcribed mRNAs from 16,780 protein-coding and 10,413 non-coding genes ( Table 2; https://rapid.ensembl.org/Ostrinia_nubilalis_GCA_963855985.1/Info/Index). The average transcript length is 13,875.99. There are 1.68 coding transcripts per gene and 5.80 exons per transcript.

Methods

Sample acquisition and DNA barcoding

An adult female specimen of Ostrinia nubilalis (specimen ID Ox000555, ToLID ilOstNubi1) was collected from Wytham Woods, Berkshire, United Kingdom (latitude 51.77, longitude –1.32) on 2020-06-25, using a light trap. The specimen was collected and identified by Douglas Boyes (University of Oxford) and preserved on dry ice.

The specimen used for Hi-C sequencing (specimen ID NHMUK014584837, ToLID ilOstNubi4) was an adult specimen collected from Lucas Road, High Wycombe, England, United Kingdom (latitude 51.63, longitude –0.74) on 2022-06-23. The specimen was collected and identified by David Lees (Natural History Museum) and preserved by dry freezing at –80 °C.

The initial identification was verified by an additional DNA barcoding process according to the framework developed by Twyford et al. (2024). A small sample was dissected from the specimens and stored in ethanol, while the remaining parts were shipped on dry ice to the Wellcome Sanger Institute (WSI). The tissue was lysed, the COI marker region was amplified by PCR, and amplicons were sequenced and compared to the BOLD database, confirming the species identification ( Crowley et al., 2023). Following whole genome sequence generation, the relevant DNA barcode region was also used alongside the initial barcoding data for sample tracking at the WSI ( Twyford et al., 2024). The standard operating procedures for Darwin Tree of Life barcoding have been deposited on protocols.io ( Beasley et al., 2023).

Nucleic acid extraction

The workflow for high molecular weight (HMW) DNA extraction at the Wellcome Sanger Institute (WSI) Tree of Life Core Laboratory includes a sequence of procedures: sample preparation and homogenisation, DNA extraction, fragmentation and purification. Detailed protocols are available on protocols.io ( Denton et al., 2023b). The ilOstNubi1 sample was prepared for DNA extraction by weighing and dissecting it on dry ice ( Jay et al., 2023). Tissue from the whole organism was homogenised using a PowerMasher II tissue disruptor ( Denton et al., 2023a).

HMW DNA was extracted using the Automated MagAttract v1 protocol ( Sheerin et al., 2023). DNA was sheared into an average fragment size of 12–20 kb in a Megaruptor 3 system ( Todorovic et al., 2023). Sheared DNA was purified by solid-phase reversible immobilisation, using AMPure PB beads to eliminate shorter fragments and concentrate the DNA ( Strickland et al., 2023). The concentration of the sheared and purified DNA was assessed using a Nanodrop spectrophotometer and a Qubit Fluorometer using the Qubit dsDNA High Sensitivity Assay kit. The fragment size distribution was evaluated by running the sample on the FemtoPulse system.

Hi-C preparation

Tissue from the ilOstNubi4 sample was processed at the WSI Scientific Operations core, using the Arima-HiC v2 kit. Tissue (stored at –80 °C) was fixed, and the DNA crosslinked using a TC buffer with 22% formaldehyde. After crosslinking, the tissue was homogenised using the Diagnocine Power Masher-II and BioMasher-II tubes and pestles. Following the kit manufacturer's instructions, crosslinked DNA was digested using a restriction enzyme master mix. The 5’-overhangs were then filled in and labelled with biotinylated nucleotides and proximally ligated. An overnight incubation was carried out for enzymes to digest remaining proteins and for crosslinks to reverse. A clean up was performed with SPRIselect beads prior to library preparation.

Library preparation and sequencing

Library preparation and sequencing were performed at the WSI Scientific Operations core. Pacific Biosciences HiFi circular consensus DNA sequencing libraries were prepared using the PacBio Express Template Preparation Kit v2.0 (Pacific Biosciences, California, USA) as per the manufacturer's instructions. The kit includes the reagents required for removal of single-strand overhangs, DNA damage repair, end repair/A-tailing, adapter ligation, and nuclease treatment. Library preparation also included a library purification step using AMPure PB beads (Pacific Biosciences, California, USA) and size selection step to remove templates shorter than 3 kb using AMPure PB modified SPRI. DNA concentration was quantified using the Qubit Fluorometer v2.0 and Qubit HS Assay Kit and the final library fragment size analysis was carried out using the Agilent Femto Pulse Automated Pulsed Field CE Instrument and 165kb gDNA and 55kb BAC analysis kit. Samples were sequenced using the Sequel IIe system (Pacific Biosciences, California, USA). The concentration of the library loaded onto the Sequel IIe was in the range 40–135 pM. The SMRT link software, a PacBio web-based end-to-end workflow manager, was used to set-up and monitor the run, as well as perform primary and secondary analysis of the data upon completion.

For Hi-C library preparation, DNA was fragmented to a size of 400 to 600 bp using a Covaris E220 sonicator. The DNA was then enriched, barcoded, and amplified using the NEBNext Ultra II DNA Library Prep Kit following manufacturers’ instructions. The Hi-C sequencing was performed using paired-end sequencing with a read length of 150 bp on an Illumina NovaSeq 6000 instrument.

Genome assembly, curation and evaluation

Assembly

The HiFi reads were first assembled using Hifiasm ( Cheng et al., 2021) with the --primary option. Haplotypic duplications were identified and removed using purge_dups ( Guan et al., 2020). The Hi-C reads were mapped to the primary contigs using bwa-mem2 ( Vasimuddin et al., 2019). The contigs were further scaffolded using the provided Hi-C data ( Rao et al., 2014) in YaHS ( Zhou et al., 2023) using the --break option for handling potential misassemblies. The scaffolded assemblies were evaluated using Gfastats ( Formenti et al., 2022), BUSCO ( Manni et al., 2021) and MERQURY.FK ( Rhie et al., 2020), using a k-mer database ( k = 31) constructed using FastK.

The mitochondrial genome was assembled using MitoHiFi ( Uliano-Silva et al., 2023), which runs MitoFinder ( Allio et al., 2020) and uses these annotations to select the final mitochondrial contig and to ensure the general quality of the sequence.

Assembly curation

The assembly was decontaminated using the Assembly Screen for Cobionts and Contaminants (ASCC) pipeline (article in preparation). Flat files and maps used in curation were generated in TreeVal ( Pointon et al., 2023). Manual curation was primarily conducted using PretextView ( Harry, 2022), with additional insights provided by JBrowse2 ( Diesh et al., 2023) and HiGlass ( Kerpedjiev et al., 2018). Scaffolds were visually inspected and corrected as described by Howe et al. (2021). Any identified contamination, missed joins, and mis-joins were corrected, and duplicate sequences were tagged and removed. The curation process is documented at https://gitlab.com/wtsi-grit/rapid-curation (article in preparation).

Assembly quality assessment

The MerquryFK tool ( Rhie et al., 2020), run within a Singularity container ( Kurtzer et al., 2017), was used to evaluate k-mer completeness and assembly quality for the curated haplotypes (Hap1/2), using the k-mer databases ( k = 31) that were pre-computed prior to genome assembly. The analysis outputs included assembly QV scores and completeness statistics.

Three Nextflow ( Di Tommaso et al., 2017) DSL2 pipelines: sanger-tol/readmapping ( Surana et al., 2023a), sanger-tol/genomenote ( Surana et al., 2023b), and sanger-tol/blobtoolkit ( Muffato et al., 2024). The readmapping pipeline aligns the Hi-C reads using bwa-mem2 ( Vasimuddin et al., 2019) and combines the alignment files with SAMtools ( Danecek et al., 2021). The genomenote pipeline converts the Hi-C alignments into a contact map using BEDTools ( Quinlan & Hall, 2010) and the Cooler tool suite ( Abdennur & Mirny, 2020). The contact map is visualised in HiGlass ( Kerpedjiev et al., 2018).

The blobtoolkit pipeline is a Nextflow port of the previous Snakemake Blobtoolkit pipeline ( Challis et al., 2020). It aligns the PacBio reads in SAMtools and minimap2 ( Li, 2018) and generates coverage tracks for regions of fixed size. In parallel, it queries the GoaT database ( Challis et al., 2023) to identify all matching BUSCO lineages to run BUSCO ( Manni et al., 2021). For the three domain-level BUSCO lineages, the pipeline aligns the BUSCO genes to the UniProt Reference Proteomes database ( Bateman et al., 2023) with DIAMOND blastp ( Buchfink et al., 2021). The genome is also divided into chunks according to the density of the BUSCO genes from the closest taxonomic lineage, and each chunk is aligned to the UniProt Reference Proteomes database using DIAMOND blastx. Genome sequences without a hit are chunked using seqtk and aligned to the NT database with blastn ( Altschul et al., 1990). The blobtools suite combines all these outputs into a blobdir for visualisation.

The genome evaluation pipelines were developed using nf-core tooling ( Ewels et al., 2020) and MultiQC ( Ewels et al., 2016), relying on the Conda package manager, the Bioconda initiative ( Grüning et al., 2018), the Biocontainers infrastructure ( da Veiga Leprevost et al., 2017), as well as the Docker ( Merkel, 2014) and Singularity ( Kurtzer et al., 2017) containerisation solutions.

Table 4 contains a list of relevant software tool versions and sources.

Table 4. Software tools: versions and sources.

Software
tool		 Version Source
BEDTools 2.30.0 https://github.com/arq5x/bedtools2
BLAST 2.14.0 http://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/
BlobToolKit 4.3.7 https://github.com/blobtoolkit/blobtoolkit
BUSCO 5.4.3 and 5.5.0 https://gitlab.com/ezlab/busco
bwa-mem2 2.2.1 https://github.com/bwa-mem2/bwa-mem2
Cooler 0.8.11 https://github.com/open2c/cooler
DIAMOND 2.1.8 https://github.com/bbuchfink/diamond
fasta_windows 0.2.4 https://github.com/tolkit/fasta_windows
FastK 427104ea91c78c3b8b8b49f1a7d6bbeaa869ba1c https://github.com/thegenemyers/FASTK
Gfastats 1.3.6 https://github.com/vgl-hub/gfastats
GoaT CLI 0.2.5 https://github.com/genomehubs/goat-cli
Hifiasm 0.19.8-r587 https://github.com/chhylp123/hifiasm
HiGlass 44086069ee7d4d3f6f3f0012569789ec138f42b84
aa44357826c0b6753eb28de		 https://github.com/higlass/higlass
Merqury.FK d00d98157618f4e8d1a9190026b19b471055b22e https://github.com/thegenemyers/MERQURY.FK
MitoHiFi 3 https://github.com/marcelauliano/MitoHiFi
MultiQC 1.14, 1.17, and 1.18 https://github.com/MultiQC/MultiQC
NCBI Datasets 15.12.0 https://github.com/ncbi/datasets
Nextflow 23.04.0-5857 https://github.com/nextflow-io/nextflow
PretextView 0.2.5 https://github.com/sanger-tol/PretextView
purge_dups 1.2.5 https://github.com/dfguan/purge_dups
samtools 1.16.1, 1.17, and 1.18 https://github.com/samtools/samtools
sanger-tol/
ascc		 - https://github.com/sanger-tol/ascc
sanger-tol/
blobtoolkit		 0.6.0 https://github.com/sanger-tol/blobtoolkit
sanger-tol/
genomenote		 1.1.1 https://github.com/sanger-tol/genomenote
sanger-tol/
readmapping		 1.2.1 https://github.com/sanger-tol/readmapping
Seqtk 1.3 https://github.com/lh3/seqtk
Singularity 3.9.0 https://github.com/sylabs/singularity
TreeVal 1.0.0 https://github.com/sanger-tol/treeval
YaHS 1.2a.2 https://github.com/c-zhou/yahs
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Genome annotation

The Ensembl Genebuild annotation system ( Aken et al., 2016) was used to generate annotation for the Ostrinia nubilalis assembly (GCA_963855985.1) in Ensembl Rapid Release at the EBI. Annotation was created primarily through alignment of transcriptomic data to the genome, with gap filling via protein-to-genome alignments of a select set of proteins from UniProt ( UniProt Consortium, 2019).

Wellcome Sanger Institute – Legal and Governance

The materials that have contributed to this genome note have been supplied by a Darwin Tree of Life Partner. The submission of materials by a Darwin Tree of Life Partner is subject to the ‘Darwin Tree of Life Project Sampling Code of Practice’, which can be found in full on the Darwin Tree of Life website here. By agreeing with and signing up to the Sampling Code of Practice, the Darwin Tree of Life Partner agrees they will meet the legal and ethical requirements and standards set out within this document in respect of all samples acquired for, and supplied to, the Darwin Tree of Life Project.

Further, the Wellcome Sanger Institute employs a process whereby due diligence is carried out proportionate to the nature of the materials themselves, and the circumstances under which they have been/are to be collected and provided for use. The purpose of this is to address and mitigate any potential legal and/or ethical implications of receipt and use of the materials as part of the research project, and to ensure that in doing so we align with best practice wherever possible. The overarching areas of consideration are:

•   Ethical review of provenance and sourcing of the material

•   Legality of collection, transfer and use (national and international)

Each transfer of samples is further undertaken according to a Research Collaboration Agreement or Material Transfer Agreement entered into by the Darwin Tree of Life Partner, Genome Research Limited (operating as the Wellcome Sanger Institute), and in some circumstances other Darwin Tree of Life collaborators.

Funding Statement

This work was supported by Wellcome through core funding to the Wellcome Sanger Institute [206194, <a href=https://doi.org/10.35802/206194>https://doi.org/10.35802/206194</a>] and the Darwin Tree of Life Discretionary Award [218328, <a href=https://doi.org/10.35802/218328>https://doi.org/10.35802/218328 </a>].

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

[version 1; peer review: 2 approved]

Data availability

European Nucleotide Archive: Ostrinia nubilalis (European corn borer). Accession number PRJEB65201; https://identifiers.org/ena.embl/PRJEB65201. The genome sequence is released openly for reuse. The Ostrinia nubilalis genome sequencing initiative is part of the Darwin Tree of Life (DToL) project. All raw sequence data and the assembly have been deposited in INSDC databases. Raw data and assembly accession identifiers are reported in Table 1 and Table 2.

Author information

Members of the University of Oxford and Wytham Woods Genome Acquisition Lab are listed here: https://doi.org/10.5281/zenodo.12157525.

Members of the Natural History Museum Genome Acquisition Lab are listed here: https://doi.org/10.5281/zenodo.12159242.

Members of the Darwin Tree of Life Barcoding collective are listed here: https://doi.org/10.5281/zenodo.12158331.

Members of the Wellcome Sanger Institute Tree of Life Management, Samples and Laboratory team are listed here: https://doi.org/10.5281/zenodo.12162482.

Members of Wellcome Sanger Institute Scientific Operations: Sequencing Operations are listed here: https://doi.org/10.5281/zenodo.12165051.

Members of the Wellcome Sanger Institute Tree of Life Core Informatics team are listed here: https://doi.org/10.5281/zenodo.12160324.

Members of the Tree of Life Core Informatics collective are listed here: https://doi.org/10.5281/zenodo.12205391.

Members of the Darwin Tree of Life Consortium are listed here: https://doi.org/10.5281/zenodo.4783558.

References

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Wellcome Open Res. 2025 Feb 24. doi: 10.21956/wellcomeopenres.25924.r117296

Reviewer response for version 1

Himanshu Dubey 1

The manuscript investigates the genome assembly of the European corn borer ( Ostrinia nubilalis), an economically significant pest species. It addresses a critical agricultural challenge by providing a comprehensive genomic resource to support future research on pest management and resistance mechanisms. The high-quality genome assembly, including chromosomal pseudomolecules and the mitochondrial genome, offers valuable insights for both functional and evolutionary studies. Overall, the manuscript is suitable for indexing.

Are sufficient details of methods and materials provided to allow replication by others?

Yes

Is the rationale for creating the dataset(s) clearly described?

Yes

Are the datasets clearly presented in a useable and accessible format?

Yes

Are the protocols appropriate and is the work technically sound?

Yes

Reviewer Expertise:

Genomics

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Wellcome Open Res. 2025 Jan 18. doi: 10.21956/wellcomeopenres.25924.r117297

Reviewer response for version 1

Arun Arumugaperumal 1, Achsha Babu 2

Background

Ostrinia nubilalis, a moth belonging to Crambidae family originally native to Europe, Western Asia, and the Mediterranean, has emerged as a significant pest in North America, resulting in annual losses of over $1 billion in cultivated maize. While transgenic maize expressing Bt proteins suppressed populations since 1995, resistance to Cry1F and Cry1Ab proteins has emerged in recent years. This sexually dimorphic species shows ecological variation, including pheromone-mediated mate selection and latitudinal differences in reproductive cycles. Genetic factors like the autosomal gene pgfar and sex-linked genes bab, per, and pdfr, along with a Z-chromosome inversion, influence these adaptations. The study uses next-generation sequencing to assemble and annotate the genome, offering insights into the evolutionary and ecological dynamics of O. nubilalis and related species.

Study Objectives

The study aims to elucidate the genetic underpinnings of O. nubilalis by sequencing its genome, which remains a significant challenge due to its complex biology. Understanding its genetics could lead to innovative pest control strategies, reducing dependence on chemical pesticides and supporting sustainable agriculture through integrated pest management (IPM).

Methodology

Initially, samples were collected from Wytham Woods and High Wycombe, UK, with DNA barcoding using the cytochrome oxidase subunit 1 (COI) marker to confirm species identification. High quality DNA was extracted, homogenized, and purified using the MagAttract protocol, followed by fragmentation to 12–20 kb and quality assessment using Qubit and Nanodrop. Hi-C preparation involved tissue fixation, DNA crosslinking, and biotinylation, with subsequent library preparation using PacBio Express Kit for HiFi libraries and NEBNext Ultra II Kit for Hi-C libraries. The genome sequencing was conducted on PacBio Sequel IIe for HiFi libraries and Illumina NovaSeq 6000 for Hi-C libraries. The assembly was performed using Hifiasm and scaffolded with Hi-C data, with the mitochondrial genome reconstructed using MitoHiFi. Assembly curation was done using ASCC pipeline and PretextView, and genome quality was evaluated using BUSCO, Merqury.FK, and k-mer analysis. Annotation was conducted through Ensembl Genebuild using transcriptome alignment and UniProt protein-to-genome mappings.

Key Findings

The genome of O. nubilalis was sequenced using Pacific Biosciences HiFi long reads, generating 21.96 Gb from 1.50 million reads, achieving 44-fold coverage. The primary assembly was scaffolded with 106.66 Gb of Hi-C data. After manual curation, including fixing 12 missing joins and 4 haplotypic duplications, the final assembly consists of 495.50 Mb in 52 scaffolds with a scaffold N50 of 16.5 Mb. The majority (99.87%) of the sequence was assigned to 32 chromosomal-level scaffolds, and the sex chromosomes were identified by coverage. The final assembly has a quality value of 68.8, with a k-mer completeness of 98.91%. BUSCO analysis indicated a completeness score of 98.5%. The genome was annotated with 45,650 transcribed mRNAs from 16,780 protein-coding and 10,413 non-coding genes, with an average transcript length of 13,875.99.

Strength of the study

Using both short-read and long-read sequencing techniques increases the assembly quality, making the results more reliable and comprehensive.

Limitation and future work

A deeper exploration of genome's complexity, especially its high proportion of repetitive sequences, may hinder transcriptomics or proteomics analysis. While the gene annotations offer theoretical insights, the lack of experimental validation limits conclusions about their roles in the biology of O. nubilalis. The comparative genomics approach deepens understanding of its evolutionary relationships with other Lepidoptera species and its ecological adaptability.

Conclusion

This research provides a valuable contribution to genomics by delivering the complete genome of O. nubilalis, laying the foundation for studying its interactions with hosts and adaptation to environmental pressures, thereby advancing sustainable pest control strategies.

Are sufficient details of methods and materials provided to allow replication by others?

Yes

Is the rationale for creating the dataset(s) clearly described?

Yes

Are the datasets clearly presented in a useable and accessible format?

Yes

Are the protocols appropriate and is the work technically sound?

Yes

Reviewer Expertise:

Bioinformatics, Genomics

We confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Data Availability Statement

    European Nucleotide Archive: Ostrinia nubilalis (European corn borer). Accession number PRJEB65201; https://identifiers.org/ena.embl/PRJEB65201. The genome sequence is released openly for reuse. The Ostrinia nubilalis genome sequencing initiative is part of the Darwin Tree of Life (DToL) project. All raw sequence data and the assembly have been deposited in INSDC databases. Raw data and assembly accession identifiers are reported in Table 1 and Table 2.

    Articles from Wellcome Open Research are provided here courtesy of The Wellcome Trust

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