Abstract
We present a genome assembly from an individual female Noctua pronuba (the large yellow underwing; Arthropoda; Insecta; Lepidoptera; Noctuidae). The genome sequence is 529 megabases in span. The complete assembly is scaffolded into 32 chromosomal pseudomolecules, with the W and Z sex chromosome assembled. The mitochondrial genome was also assembled and is 15.3 kilobases in length.
Keywords: Noctua pronuba, large yellow underwing, genome sequence, chromosomal, Lepidoptera
Species taxonomy
Eukaryota; Metazoa; Ecdysozoa; Arthropoda; Hexapoda; Insecta; Pterygota; Neoptera; Endopterygota; Lepidoptera; Glossata; Ditrysia; Noctuoidea; Noctuidae; Noctuinae; Noctuini; Noctua; Noctua fimbriata (Linnaeus, 1758) (NCBI:txid214277).
Background
Noctua pronuba (large yellow underwing) is a widespread noctuid moth found across Eurasia and North Africa, and one of the most familiar moths in the UK with hundreds of adults commonly caught in a single light trap. The larvae are polyphagous, feeding on a wide range of grasses and herbaceous plants, and have bright green colouration in early instars, changing to brown in later instars. The moth was recorded in Canada in 1979 and has spread south since its accidental introduction; N. pronuba is now an occasional pest of commercial crops in the United States, where the larvae are known as winter cutworm ( Difonzo & Russell, 2010). In a major outbreak in Michigan in 2007, vast numbers of larvae caused defoliation of fields of alfalfa, rye, oat and winter wheat, and reached public nuisance proportions ( Difonzo & Russell, 2010).
The forewings of the adult moth may be light brown, ochreous or dark-purplish brown, with colour controlled genetically through polymorphism at unknown loci modified by sex-linked genes ( Cook & Sarsam, 1981). The hindwings, which are completely hidden at rest, are bright orange-yellow with a black band. When disturbed and the moth takes flight, the yellow hindwings are suddenly revealed, plausibly acting as ‘flash colouration’ to startle predators. In Europe, N. pronuba is a strongly migratory species: vertical-looking entomological radars sited in the UK have detected large numbers of individuals flying north in spring or south in autumn ( Chapman et al., 2010). Indeed, in experiments using moths tethered to a flight mill N. pronuba was found to be one of the most mobile noctuid species as measured by both maximum flight speed and total distance covered in one night ( Jones et al., 2015). Migration direction is thought to be affected by use of magnetic compass sense, coupled with the ability to detect and utilise favourably-directed winds ( Baker & Mather, 1982; Reynolds et al., 2010).
The genome of N. pronuba was sequenced as part of the Darwin Tree of Life Project, a collaborative effort to sequence all of the named eukaryotic species in the Atlantic Archipelago of Britain and Ireland. Here we present a chromosomally complete genome sequence for N. pronuba, based on one female specimen from Wytham Woods, Oxfordshire, UK.
Genome sequence report
The genome was sequenced from a single female N. pronuba collected from Wytham Woods, Oxfordshire, UK (latitude 51.772, longitude -1.338) ( Figure 1). A total of 47-fold coverage in Pacific Biosciences single-molecule HiFi long reads and 71-fold coverage in 10X Genomics read clouds were generated. Primary assembly contigs were scaffolded with chromosome conformation Hi-C data. Manual assembly curation corrected 21 missing/misjoins and removed 6 haplotypic duplications, reducing the assembly length by 0.02% and the scaffold number by 25.76%, and increasing the scaffold N50 by 1.57%.
Figure 1. Image of the ilNocPron1 specimen taken during preservation and processing.
The final assembly has a total length of 529 Mb in 49 sequence scaffolds with a scaffold N50 of 17.9 Mb ( Table 1). Of the assembly sequence, 99.89% was assigned to 32 chromosomal-level scaffolds, representing 30 autosomes (numbered by sequence length), and the W and Z sex chromosome ( Figure 2– Figure 5; Table 2). The assembly has a BUSCO ( Manni et al., 2021) completeness of 98.9% using the lepidoptera_odb10 reference set. While not fully phased, the assembly deposited is of one haplotype. Contigs corresponding to the second haplotype have also been deposited.
Figure 2. Genome assembly of Noctua pronuba, ilNocPron1.1: metrics.
The BlobToolKit Snailplot shows N50 metrics and BUSCO gene completeness. The main plot is divided into 1,000 size-ordered bins around the circumference with each bin representing 0.1% of the 529,194,529 bp assembly. The distribution of chromosome lengths is shown in dark grey with the plot radius scaled to the longest chromosome present in the assembly (24,140,489 bp, shown in red). Orange and pale-orange arcs show the N50 and N90 chromosome lengths (17,891,575 and 12,542,342 bp), respectively. The pale grey spiral shows the cumulative chromosome count on a log scale with white scale lines showing successive orders of magnitude. The blue and pale-blue area around the outside of the plot shows the distribution of GC, AT and N percentages in the same bins as the inner plot. A summary of complete, fragmented, duplicated and missing BUSCO genes in the lepidoptera_odb10 set is shown in the top right. An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/ilNocPron1.1/dataset/CAJMZM01/snail.
Figure 5. Genome assembly of Noctua pronuba, ilNocPron1.1: Hi-C contact map.
Hi-C contact map of the ilNocPron1.1 assembly, visualised in HiGlass. Chromosomes are shown in order of size from left to right and top to bottom.
Table 1. Genome data for Noctua pronuba, ilNocPron1.1.
| Project accession data | |
|---|---|
| Assembly identifier | ilNocPron1.1 |
| Species | Noctua pronuba |
| Specimen | ilNocPron1 |
| NCBI taxonomy ID | NCBI:txid214277 |
| BioProject | PRJEB43815 |
| BioSample ID | SAMEA7519837 |
| Isolate information | Female, whole organism |
| Raw data accessions | |
| PacificBiosciences SEQUEL II | ERR6590587 |
| 10X Genomics Illumina | ERR6054676-ERR6054679 |
| Hi-C Illumina | ERR6054680-ERR6054682 |
| Genome assembly | |
| Assembly accession | GCA_905163415.1 |
| Accession of alternate haplotype | GCA_905220345.1 |
| Span (Mb) | 529 |
| Number of contigs | 74 |
| Contig N50 length (Mb) | 16.2 |
| Number of scaffolds | 49 |
| Scaffold N50 length (Mb) | 17.9 |
| Longest scaffold (Mb) | 21.7 |
| BUSCO * genome score | C:98.9%[S:98.3%,D:0.5%],
F:0.2%,M:0.9%,n:5286 |
*BUSCO scores based on the lepidoptera_odb10 BUSCO set using v5.1.2. 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/ilNocPron1.1/dataset/CAJMZM01/busco
Figure 3. Genome assembly of Noctua pronuba, ilNocPron1.1: GC coverage.
BlobToolKit GC-coverage plot. Scaffolds are coloured by phylum. Circles are sized in proportion to scaffold length. Histograms show the distribution of scaffold length sum along each axis. An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/ilNocPron1.1/dataset/CAJMZM01/blob.
Figure 4. Genome assembly of Noctua pronuba, ilNocPron1.1: cumulative sequence.
BlobToolKit cumulative sequence plot. 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/ilNocPron1.1/dataset/CAJMZM01/cumulative.
Table 2. Chromosomal pseudomolecules in the genome assembly of Noctua pronuba, ilNocPron1.1.
| INSDC
accession |
Chromosome | Size (Mb) | GC% |
|---|---|---|---|
| LR999893.1 | 1 | 20.39 | 38.0 |
| LR999894.1 | 2 | 19.95 | 38.2 |
| LR999895.1 | 3 | 19.85 | 37.6 |
| LR999896.1 | 4 | 19.06 | 37.7 |
| LR999897.1 | 5 | 18.68 | 38.2 |
| LR999898.1 | 6 | 18.65 | 37.8 |
| LR999899.1 | 7 | 18.55 | 37.7 |
| LR999900.1 | 8 | 18.48 | 38.1 |
| LR999901.1 | 9 | 18.20 | 38.2 |
| LR999902.1 | 10 | 18.14 | 37.8 |
| LR999903.1 | 11 | 17.91 | 37.8 |
| LR999904.1 | 12 | 17.89 | 37.9 |
| LR999905.1 | 13 | 17.62 | 38.0 |
| LR999906.1 | 14 | 17.46 | 38.2 |
| LR999907.1 | 15 | 17.25 | 37.9 |
| LR999908.1 | 16 | 17.15 | 38.1 |
| LR999909.1 | 17 | 17.08 | 38.3 |
| LR999910.1 | 18 | 16.84 | 38.0 |
| LR999911.1 | 19 | 16.20 | 38.4 |
| LR999912.1 | 20 | 15.88 | 38.5 |
| LR999913.1 | 21 | 15.49 | 38.6 |
| LR999914.1 | 22 | 15.37 | 38.0 |
| LR999915.1 | 23 | 14.84 | 38.2 |
| LR999916.1 | 24 | 14.16 | 38.7 |
| LR999917.1 | 25 | 12.54 | 38.3 |
| LR999918.1 | 26 | 12.31 | 38.4 |
| LR999919.1 | 27 | 10.04 | 38.8 |
| LR999920.1 | 28 | 9.48 | 38.8 |
| LR999921.1 | 29 | 9.39 | 39.0 |
| LR999922.1 | 30 | 9.09 | 39.8 |
| LR999892.1 | W | 20.55 | 40.1 |
| LR999891.1 | Z | 24.14 | 37.8 |
| LR999923.1 | MT | 0.02 | 18.9 |
| - | Unplaced | 0.55 | 45.4 |
Methods
Sample acquisition, DNA extraction and sequencing
A single female N. pronuba (ilNocPron1) was collected from Wytham Woods, Oxfordshire, UK (latitude 51.772, longitude -1.338) by Douglas Boyes, UKCEH, and identified by the same individual. The specimen was collected using a light trap in woodland, preserved on dry ice prior to transfer to the Wellcome Sanger Institute.
DNA was extracted from whole organism tissue at the Wellcome Sanger Institute (WSI) Scientific Operations core from the whole organism using the Qiagen MagAttract HMW DNA kit, according to the manufacturer’s instructions. Pacific Biosciences HiFi circular consensus and 10X Genomics read cloud sequencing libraries were constructed according to the manufacturers’ instructions. Sequencing was performed by the Scientific Operations core at the Wellcome Sanger Institute on Pacific Biosciences SEQUEL II and Illumina HiSeq X instruments. Hi-C data were generated from remaining whole organism tissue using the Arima Hi-C v2 kit and sequenced on HiSeq X.
Genome assembly
Assembly was carried out with HiCanu ( Nurk et al., 2020); haplotypic duplication was identified and removed with purge_dups ( Guan et al., 2020). One round of polishing was performed by aligning 10X Genomics read data to the assembly with longranger align, calling variants with freebayes ( Garrison & Marth, 2012). The assembly was then scaffolded with Hi-C data ( Rao et al., 2014) using SALSA2 ( Ghurye et al., 2019). The assembly was checked for contamination and corrected using the gEVAL system ( Chow et al., 2016) as described previously ( Howe et al., 2021). Manual curation ( Howe et al., 2021) was performed using gEVAL, HiGlass ( Kerpedjiev et al., 2018) and Pretext. The mitochondrial genome was assembled using MitoHiFi ( Uliano-Silva et al., 2021), which performed annotation using MitoFinder ( Allio et al., 2020). The genome was analysed and BUSCO scores generated within the BlobToolKit environment ( Challis et al., 2020). Table 3 contains a list of all software tool versions used, where appropriate.
Table 3. Software tools used.
| Software tool | Version | Source |
|---|---|---|
| HiCanu | 2.1 | Nurk et al., 2020 |
| purge_dups | 1.2.3 | Guan et al., 2020 |
| SALSA2 | 2.2 | Ghurye et al., 2019 |
| longranger align | 2.2.2 |
https://support.10xgenomics.com/genome-exome/
software/pipelines/latest/advanced/other-pipelines |
| freebayes | 1.3.1-17-gaa2ace8 | Garrison & Marth, 2012 |
| MitoHiFi | 1 | Uliano-Silva et al., 2021 |
| gEVAL | N/A | Chow et al., 2016 |
| PretextView | 0.1.x | https://github.com/wtsi-hpag/PretextView |
| HiGlass | 1.11.6 | Kerpedjiev et al., 2018 |
| BlobToolKit | 2.6.4 | Challis et al., 2020 |
Ethics/compliance issues
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. 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. 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.
Data availability
European Nucleotide Archive: Noctua pronuba (large yellow underwing). Accession number PRJEB43815; https://identifiers.org/ena.embl/PRJEB43815.
The genome sequence is released openly for reuse. The N. pronuba 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. The genome will be annotated and presented through the Ensembl pipeline at the European Bioinformatics Institute. Raw data and assembly accession identifiers are reported in Table 1.
Author information
Members of the University of Oxford and Wytham Woods Genome Acquisition Lab are listed here: https://doi.org/10.5281/zenodo.5746938.
Members of the Darwin Tree of Life Barcoding collective are listed here: https://doi.org/10.5281/zenodo.5744972.
Members of the Wellcome Sanger Institute Tree of Life programme are listed here: https://doi.org/10.5281/zenodo.6125027.
Members of Wellcome Sanger Institute Scientific Operations: DNA Pipelines collective are listed here: https://doi.org/10.5281/zenodo.5746904.
Members of the Tree of Life Core Informatics collective are listed here: https://doi.org/10.5281/zenodo.6125046.
Members of the Darwin Tree of Life Consortium are listed here: https://doi.org/10.5281/zenodo.5638618.
Funding Statement
This work was supported by Wellcome through core funding to the Wellcome Sanger Institute (206194) and the Darwin Tree of Life Discretionary Award (218328).
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]
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