Abstract
We present a genome assembly from an individual male Xestia c-nigrum (the setaceous Hebrew character; Arthropoda; Insecta; Lepidoptera; Noctuidae). The genome sequence is 760 megabases in span. Most of the assembly is scaffolded into 31 chromosomal pseudomolecules, including the assembled Z sex chromosome. The mitochondrial genome has also been assembled and is 15.3 kilobases in length.
Keywords: Xestia c-nigrum, setaceous Hebrew character, genome sequence, chromosomal, Lepidoptera
Species taxonomy
Eukaryota; Metazoa; Ecdysozoa; Arthropoda; Hexapoda; Insecta; Pterygota; Neoptera; Endopterygota; Lepidoptera; Glossata; Ditrysia; Noctuoidea; Noctuidae; Noctuinae; Noctuini; Xestia; Xestia c-nigrum (Linnaeus, 1758) (NCBI:txid987431).
Background
Known to most British lepidopterists as setaceous Hebrew character, Xestia c-nigrum is generally referred to as the spotted cutworm in the pest control literature. The latter name is derived from the appearance of the caterpillar while ‘ c-nigrum’ and ‘Hebrew character’ reference the distinctive black marking on the forewing.
X. c-nigrum is a familiar, widespread species across Asia, Europe and North America. In much of its range, including Britain, there are two generations in a year, the second usually much larger. The summer and autumn cohort might be larger due to increased survival (larvae of the spring cohort over-winter) and/or immigration from further south ( Clancy et al., 2012). Larvae feed on a variety of herbaceous plants and over-winter as diapausing larvae, growing at a slower rate than non-diapausing larvae ( Honek, 1979). A granulovirus isolated from X. c-nigrum has been used to develop virus-based biopesticides against agricultural pest moths, e.g. ( Goto et al., 2015).
This is the second whole genome sequence for a Xestia moth species, following that of X. xanthographa ( Boyes & Holland, 2022). The sequenced individual was collected at Hever Castle during a collecting trip for the Natural History Museum Darwin Tree of Life sampling team.
Genome sequence report
The genome was sequenced from one male X. c-nigrum ( Figure 1) collected from Hever Castle, England, UK (latitude 51.188, longitude 0.12). A total of 35-fold coverage in Pacific Biosciences single-molecule HiFi long reads and 58-fold coverage in 10X Genomics read clouds were generated. Primary assembly contigs were scaffolded with chromosome conformation Hi-C data. Manual assembly curation corrected nine (9) missing/misjoins, reducing the assembly length by 0.03% and the scaffold number by 14%, and increasing the scaffold N50 by 0.1%.
Figure 1. Image of the Xestia c-nigrum (ilXesCnig1) specimen used for genome sequencing.
The final assembly has a total length of 760 Mb in 43 sequence scaffolds with a scaffold N50 of 25.7 Mb ( Table 1). Most of the assembly sequence (99.9%) was assigned to 31 chromosomal-level scaffolds confirmed by Hi-C data, representing 30 autosomes named in order of size and the Z chromosome ( Figure 2– Figure 5, Table 2). The assembly has a BUSCO 5.3.2 ( Manni et al., 2021) completeness of 98.8% using the lepidoptera_odb10 reference set.
Figure 2. Genome assembly of Xestia c-nigrum, ilXesCnig1.1: 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 760,318,956 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 (43,326,432 bp, shown in red). Orange and pale-orange arcs show the N50 and N90 chromosome lengths (25,717,762 and 18,159,168 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/ilXesCnig1.1/dataset/CAKAJW01.1/snail.
Figure 5. Genome assembly of Xestia c-nigrum (ilXesCnig1.1): Hi-C contact map.
Hi-C contact map of the ilXesCnig1.1 assembly, visualised using HiGlass. Chromosomes are given in order of size from left to right and top to bottom. The interactive Hi-C map can be viewed at https://genome-note-higlass.tol.sanger.ac.uk/l/?d=CB8TqIaYQNqsG4GcF9YwLg.
Table 1. Genome data for Xestia c-nigrum (ilXesCnig1.1).
| Project accession data | |
|---|---|
| Assembly identifier | ilXesCnig1.1 |
| Species | Xestia c-nigrum |
| Specimen | ilXesCnig1 (genome assembly,
Hi-C), ilXesCnig2 (RNA-Seq) |
| NCBI taxonomy ID | 987431 |
| BioProject | PRJEB46327 |
| BioSample ID | SAMEA8239458 |
| Isolate information | Male, whole organism
(ilXesCnig1); undescribed, whole organism (ilXesCnig2) |
| Raw data accessions | |
| PacificBiosciences SEQUEL II | ERR6939245, ERR6939246 |
| 10X Genomics Illumina | ERR6688565–ERR6688568 |
| Hi-C Illumina | ERR6688569 |
| PolyA RNA-Seq Illumina | ERR9435011 |
| Genome assembly | |
| Assembly accession | GCA_916618015.1 |
| Accession of alternate haplotype | GCA_916617455.1 |
| Span (Mb) | 760 |
| Number of contigs | 60 |
| Contig N50 length (Mb) | 25.0 |
| Number of scaffolds | 43 |
| Scaffold N50 length (Mb) | 25.7 |
| Longest scaffold (Mb) | 29.1 |
| BUSCO * genome score | C:98.8%[S:98.2%,D:0.5%],
F:0.2%,M:1.0%,n:5,286 |
* BUSCO scores based on the lepidoptera_odb1 BUSCO set using v5.3.2. 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/ilXesCnig1.1/dataset/CAKAJW01.1/busco.
Figure 3. Genome assembly of Xestia c-nigrum, ilXesCnig1.1: GC coverage.
BlobToolKit GC-coverage plot. Chromosomes are coloured by phylum. Circles are sized in proportion to chromosome length. Histograms show the distribution of chromosome length sum along each axis. An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/ilXesCnig1.1/dataset/CAKAJW01.1/blob.
Figure 4. Genome assembly of Xestia c-nigrum (ilXesCnig1.1): cumulative sequence.
BlobToolKit cumulative sequence plot. The grey line shows cumulative length for all chromosomes. Coloured lines show cumulative lengths of chromosomes assigned to each phylum using the buscogenes taxrule. An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/ilXesCnig1.1/dataset/CAKAJW01.1/cumulative.
Table 2. Chromosomal pseudomolecules in the genome assembly of Xestia c-nigrum, ilXesCnig1.
| INSDC accession | Chromosome | Size (Mb) | GC% |
|---|---|---|---|
| OU745244.1 | 1 | 29.1 | 38.2 |
| OU745245.1 | 2 | 28.86 | 38.4 |
| OU745246.1 | 3 | 28.59 | 38.4 |
| OU745247.1 | 4 | 28.48 | 38.3 |
| OU745248.1 | 5 | 27.79 | 38.4 |
| OU745249.1 | 6 | 26.8 | 38.3 |
| OU745250.1 | 7 | 26.79 | 37.9 |
| OU745251.1 | 8 | 26.44 | 38.6 |
| OU745252.1 | 9 | 26.25 | 38.2 |
| OU745253.1 | 10 | 26.15 | 38.1 |
| OU745254.1 | 11 | 26.05 | 38.6 |
| OU745255.1 | 12 | 26 | 38.5 |
| OU745256.1 | 13 | 25.72 | 38.3 |
| OU745257.1 | 14 | 25.7 | 37.9 |
| OU745258.1 | 15 | 25.09 | 38.4 |
| OU745259.1 | 16 | 25.04 | 38.5 |
| OU745260.1 | 17 | 24.44 | 38.4 |
| OU745261.1 | 18 | 23.84 | 38.5 |
| OU745262.1 | 19 | 23.83 | 38.3 |
| OU745263.1 | 20 | 23.45 | 38.6 |
| OU745264.1 | 21 | 23.03 | 38 |
| OU745265.1 | 22 | 22.35 | 38.7 |
| OU745266.1 | 23 | 22.32 | 38.5 |
| OU745267.1 | 24 | 21.62 | 38.9 |
| OU745268.1 | 25 | 21.11 | 38.7 |
| OU745269.1 | 26 | 18.16 | 38.5 |
| OU745270.1 | 27 | 17.02 | 38.5 |
| OU745271.1 | 28 | 16.11 | 38.7 |
| OU745272.1 | 29 | 15.52 | 39.7 |
| OU745273.1 | 30 | 14.63 | 39 |
| OU745243.1 | Z | 43.33 | 38.1 |
| OU745274.1 | MT | 0.02 | 19 |
| - | unplaced | 0.7 | 48 |
While not fully phased, the assembly deposited is of one haplotype. Contigs corresponding to the second haplotype have also been deposited.
Methods
Sample acquisition and nucleic acid extraction
A male X. c-nigrum (ilXesCnig1) was collected using a light trap and identified by Gavin Broad (Natural History Museum) from Hever Castle, United Kingdom (latitude 51.188, longitude 0.12). The sample was preserved on dry ice by Laura Sivess. A second X. c nigrum (ilXesCnig2) was collected using a light trap and identified by Douglas Boyes (Natural History Museum) from Wytham Woods, United Kingdom (latitude 51.772, longitude –1.338).
DNA was extracted from head and thorax tissue of ilXesCnig1 at the Wellcome Sanger Institute (WSI) Scientific Operations core using the Qiagen MagAttract HMW DNA kit, according to the manufacturer’s instructions. RNA was extracted from abdomen tissue of ilXesCnig2 in the Tree of Life Laboratory at the WSI using TRIzol, according to the manufacturer’s instructions. RNA was then eluted in 50 μl RNAse-free water and its concentration assessed using a Nanodrop spectrophotometer and Qubit Fluorometer using the Qubit RNA Broad-Range (BR) Assay kit. Analysis of the integrity of the RNA was done using Agilent RNA 6000 Pico Kit and Eukaryotic Total RNA assay.
Sequencing
Pacific Biosciences HiFi circular consensus and 10X Genomics read cloud DNA sequencing libraries were constructed according to the manufacturers’ instructions. Poly(A) RNA-Seq libraries were constructed using the NEB Ultra II RNA Library Prep kit. DNA and RNA sequencing was performed by the Scientific Operations core at the WSI on Pacific Biosciences SEQUEL II (HiFi), Illumina HiSeq 4000 (RNA-Seq) and Illumina NovaSeq 6000 (10X) instruments. Hi-C data were also generated from head tissue of ilXesCnig1 using the Arima v2 kit and sequenced on the Illumina NovaSeq 6000 instrument.
Genome assembly
Assembly was carried out with Hifiasm ( Cheng et al., 2021) and 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). Manual curation ( Howe et al., 2021) was performed using HiGlass ( Kerpedjiev et al., 2018) and Pretext ( Harry, 2022). Chromosome-scale scaffolds confirmed by the Hi-C data have been named in order of size. The mitochondrial genome was assembled using MitoHiFi ( Uliano-Silva et al., 2021), which performs annotation using MitoFinder ( Allio et al., 2020). The genome was analysed and BUSCO scores were 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 |
|---|---|---|
| BlobToolKit | 3.2.6 | ( Challis et al., 2020) |
| freebayes | v1.3.1-17-gaa2ace8 | ( Garrison & Marth, 2012) |
| hifiasm | 0.15.3 | ( Cheng et al., 2021) |
| HiGlass | 1.11.6 | ( Kerpedjiev et al., 2018) |
| longranger | 2.2.2 | https://support.10xgenomics.com/genome-exome/software/pipelines/latest/advanced/other-pipelines |
| MitoHiFi | 2.0 | ( Uliano-Silva et al., 2021) |
| PretextView | 0.2.x | https://github.com/wtsi-hpag/PretextView |
| purge_dups | 1.2.3 | ( Guan et al., 2020) |
| SALSA | 2.2 | ( Ghurye et al., 2019) |
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: Xestia c-nigrum (spotted cutworm). Accession number PRJEB46327. https://identifiers.org/ena.embl/PRJEB46327 ( Wellcome Sanger Institute, 2022).
The genome sequence is released openly for reuse. The Xestia c-nigrum 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 using available RNA-Seq data 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 Natural History Museum Genome Acquisition Lab are listed here: https://doi.org/10.5281/zenodo.4790042.
Members of the University of Oxford and Wytham Woods Genome Acquisition Lab are listed here: https://doi.org/10.5281/zenodo.4789928.
Members of the Darwin Tree of Life Barcoding collective are listed here: https://doi.org/10.5281/zenodo.4893703.
Members of the Wellcome Sanger Institute Tree of Life programme are listed here: https://doi.org/10.5281/zenodo.4783585.
Members of Wellcome Sanger Institute Scientific Operations: DNA Pipelines collective are listed here: https://doi.org/10.5281/zenodo.4790455.
Members of the Tree of Life Core Informatics collective are listed here: https://doi.org/10.5281/zenodo.5013541.
Members of the Darwin Tree of Life Consortium are listed here: https://doi.org/10.5281/zenodo.4783558.
References
- Allio R, Schomaker-Bastos A, Romiguier J, et al. : MitoFinder: Efficient automated large-scale extraction of mitogenomic data in target enrichment phylogenomics. Mol Ecol Resour. 2020;20(4):892–905. 10.1111/1755-0998.13160 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Boyes D, Holland PWH: The genome sequence of the square-spot rustic, Xestia xanthographa (Schiffermuller, 1775) [version 1; peer review: 2 approved]. Wellcome Open Res. 2022;7:37. 10.12688/wellcomeopenres.17538.1 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Challis R, Richards E, Rajan J, et al. : BlobToolKit - Interactive Quality Assessment of Genome Assemblies. G3 (Bethesda). 2020;10(4):1361–1374. 10.1534/g3.119.400908 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cheng H, Concepcion GT, Feng X, et al. : Haplotype-resolved de novo assembly using phased assembly graphs with hifiasm. Nat Methods. 2021;18(2):170–175. 10.1038/s41592-020-01056-5 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Clancy S, Top-Jensen M, Fibiger M: Moths of Great Britain and Ireland – A field guide to all the macromoths.Oestermarie, Denmark: Bugbook Publishing. 2012. Reference Source [Google Scholar]
- Garrison E, Marth G: Haplotype-based variant detection from short-read sequencing. 2012. 10.48550/arXiv.1207.3907 [DOI] [Google Scholar]
- Ghurye J, Rhie A, Walenz BP, et al. : Integrating Hi-C links with assembly graphs for chromosome-scale assembly. PLoS Comput Biol. 2019;15(8):e1007273. 10.1371/journal.pcbi.1007273 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Goto C, Mukawa S, Mitsunaga T: Two Year Field Study to Evaluate the Efficacy of Mamestra brassicae Nucleopolyhedrovirus Combined with Proteins Derived from Xestia c-nigrum Granulovirus. Viruses. 2015;7(3):1062–1078. 10.3390/v7031062 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Guan D, McCarthy SA, Wood J, et al. : Identifying and removing haplotypic duplication in primary genome assemblies. Bioinformatics. 2020;36(9):2896–2898. 10.1093/bioinformatics/btaa025 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Harry E: PretextView (Paired REad TEXTure Viewer): A desktop application for viewing pretext contact maps. 2022; (Accessed: 19 October 2022). Reference Source [Google Scholar]
- Honek A: Regulation of diapause, number of instars, and body growth in the moth species Amathes c-nigrum (Lepidoptera: Noctuidae). Entomologia Generalis. 1979;5(3):221–229. Reference Source [Google Scholar]
- Howe K, Chow W, Collins J, et al. : Significantly improving the quality of genome assemblies through curation. GigaScience. Oxford University Press,2021;10( 1):giaa153. 10.1093/gigascience/giaa153 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kerpedjiev P, Abdennur N, Lekschas F, et al. : HiGlass: Web-based visual exploration and analysis of genome interaction maps. Genome Biol. 2018;19(1):125. 10.1186/s13059-018-1486-1 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Manni M, Berkeley MR, Seppey M, et al. : BUSCO Update: Novel and Streamlined Workflows along with Broader and Deeper Phylogenetic Coverage for Scoring of Eukaryotic, Prokaryotic, and Viral Genomes. Mol Biol Evol. 2021;38(10):4647–4654. 10.1093/molbev/msab199 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rao SS, Huntley MH, Durand NC, et al. : A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping. Cell. 2014;159(7):1665–1680. 10.1016/j.cell.2014.11.021 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Uliano-Silva M, et al. : MitoHiFi. 2021; (Accessed: 19 October 2022). Reference Source [Google Scholar]
- Wellcome Sanger Institute: The genome sequence of the setaceous Hebrew character, Xestia c-nigrum, (Linnaeus, 1758), European Nucleotide Archive [dataset].accession number PRJEB46327, 2022. [DOI] [PMC free article] [PubMed]