Abstract
We present a genome assembly from an individual female Hecatera dysodea (the Small Ranunculus; Arthropoda; Insecta; Lepidoptera; Noctuidae). The genome sequence is 640.9 megabases in span. Most of the assembly is scaffolded into 32 chromosomal pseudomolecules, including the Z and W sex chromosomes. The mitochondrial genome has also been assembled and is 15.4 kilobases in length. Gene annotation of this assembly on Ensembl has identified 12,213 protein coding genes.
Keywords: Hecatera dysodea, Small Ranunculus, genome sequence, chromosomal, Lepidoptera
Species taxonomy
Eukaryota; Metazoa; Ecdysozoa; Arthropoda; Hexapoda; Insecta; Pterygota; Neoptera; Endopterygota; Lepidoptera; Glossata; Ditrysia; Noctuoidea; Noctuidae; Hadeninae; Hecatera; Hecatera dysodea (Denis & Schiffermüller, 1775) (NCBI:txid988125).
Background
Hecatera dysodea, known as the Small Ranunculus, is a moth with subtly attractive markings as an adult, and an interesting history of extinction and colonisation in the British Isles. Larvae feed on the seeds or flowers of lettuces, mainly Prickly Lettuce ( Lactuca serriola), but also other lettuce species, including cultivated varieties. Although H. dysodea has been reported as a pest of lettuces, as a seed and flower eater, they would only ever be eating bolted lettuces and thus a potential pest of lettuce seed crops. Adult moths visit flowers, especially of lettuces, and are readily attracted to light. Although it is sometimes reported as having one generation per year, Clancy et al., 2012 report two overlapping generations, with adults on the wing from May to October.
Found naturally across mainly Central and Southern Europe and Central Asia, H. dysodea has also been accidentally introduced to the USA, where it is now widespread in the Pacific Northwest ( Landolt et al., 2010). The species name ‘dysodea’ is thought to originate from the larvae, ‘ill-smelling’ ( Pratt, 1986), maybe a reference to the smell of the lettuce (they are not tasty after bolting). Chemical attractants are being used in the US to monitor and potentially control populations ( Landolt et al., 2017).
The population of H. dysodea in England was always rather cyclical, with its heyday apparently around the end of the 19th century; thereafter there was a rapid decline with extinction in this country around the 1930s. Pratt (1986) summarised the history of the decline and loss of H. dysodea from Britain and suggested that more modern farming (i.e., fewer bolting lettuce plants), declines in market gardens in south-east England and a succession of wet summers could have combined to cause its local extinction. In 1997, moths were found again in Kent ( Agassiz & Spice, 1998) and H. dysodea has rapidly spread since then, now being rather widespread in England and in parts of Wales and its British population is classified as being of Least Concern ( Fox et al., 2019). It is a species of rough, open ground, including brownfield sites and gardens.
Genome sequence report
The genome was sequenced from one female Hecatera dysodea specimen ( Figure 1) collected from Tonbridge, UK (latitude 51.186305, longitude 0.286464). A total of 47-fold coverage in Pacific Biosciences single-molecule HiFi long reads and 60-fold coverage in 10X Genomics read clouds were generated. Primary assembly contigs were scaffolded with chromosome conformation Hi-C data. Manual assembly curation corrected 70 missing or mis-joins and removed six haplotypic duplications, reducing the assembly length by 0.55%% and the scaffold number by 47.37%, and increasing the scaffold N50 by 4.54%.
Figure 1. Photograph of the Hecatera dysodea (ilHecDyso1) specimen used for genome sequencing.
The final assembly has a total length of 640.9 Mb in 40 sequence scaffolds with a scaffold N50 of 21.9 Mb ( Table 1). Most (99.94%) of the assembly sequence was assigned to 32 chromosomal-level scaffolds, representing 30 autosomes and the W and Z sex chromosomes. Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size ( Figure 2– Figure 5; Table 2). The assembly has a BUSCO v5.3.2 ( Manni et al., 2021) completeness of 99.0% (single 98.4%, duplicated 0.6%) using the lepidoptera_odb10 reference set.
Figure 2. Genome assembly of Hecatera dysodea, ilHecDyso1.2: 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 640,911,623 bp assembly. The distribution of scaffold lengths is shown in dark grey with the plot radius scaled to the longest scaffold present in the assembly (30,380,561 bp, shown in red). Orange and pale-orange arcs show the N50 and N90 scaffold lengths (21,896,367 and 15,320,587 bp), respectively. The pale grey spiral shows the cumulative scaffold 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/ilHecDyso1.2/dataset/CAJOST02/snail.
Figure 5. Genome assembly of Hecatera dysodea, ilHecDyso1.2: Hi-C contact map.
Hi-C contact map of the ilHecDyso1.2 assembly, visualised using HiGlass. 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=eT_0h2ElSVOAkIDCfeUuNA.
Table 1. Genome data for Hecatera dysodea, ilHecDyso1.2.
| Project accession data | ||
|---|---|---|
| Assembly identifier | ilHecDyso1.2 | |
| Species | Hecatera dysodea | |
| Specimen | ilHecDyso1 | |
| NCBI taxonomy ID | 988125 | |
| BioProject | PRJEB43532 | |
| BioSample ID | SAMEA7521514 | |
| Isolate information | ilHecDyso1, female | |
| Assembly metrics * | Benchmark | |
| Consensus quality (QV) | 54 | ≥ 50 |
| k-mer completeness | 99.99% | ≥ 95% |
| BUSCO ** | C:99.0%[S:98.4%,D:0.6%],
F:0.2%,M:0.8%,n:5,286 |
C ≥ 95% |
| Percentage of assembly mapped to chromosomes | 99.94% | ≥ 95% |
| Sex chromosomes | W and Z chromosomes | localised homologous pairs |
| Organelles | Mitochondrial genome assembled | complete single alleles |
| Raw data accessions | ||
| PacificBiosciences SEQUEL II | ERR6406206 | |
| 10X Genomics Illumina | ERR6054504–ERR6054507 | |
| Hi-C Illumina | ERR6054503 | |
| PolyA RNA-Seq Illumina | ERR6464926 | |
| Genome assembly | ||
| Assembly accession | GCA_905332915.2 | |
| Span (Mb) | 640.9 | |
| Number of contigs | 115 | |
| Contig N50 length (Mb) | 18.0 | |
| Number of scaffolds | 40 | |
| Scaffold N50 length (Mb) | 21.9 | |
| Longest scaffold (Mb) | 30.4 | |
| Genome annotation | ||
| Number of protein-coding genes | 12,213 | |
| Number of non-coding genes | 1,652 | |
| Number of gene transcripts | 21,454 | |
* Assembly metric benchmarks are adapted from column VGP-2020 of “Table 1: Proposed standards and metrics for defining genome assembly quality” from ( Rhie et al., 2021).
** BUSCO scores based on the lepidoptera_odb10 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/ilHecDyso1.2/dataset/CAJOST02/busco.
Figure 3. Genome assembly of Hecatera dysodea, ilHecDyso1.2: 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/ilHecDyso1.2/dataset/CAJOST02/blob.
Figure 4. Genome assembly of Hecatera dysodea, ilHecDyso1.2: 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/ilHecDyso1.2/dataset/CAJOST02/cumulative.
Table 2. Chromosomal pseudomolecules in the genome assembly of Hecatera dysodea, ilHecDyso1.
| INSDC accession | Chromosome | Size (Mb) | GC% |
|---|---|---|---|
| HG995287.1 | 1 | 26.17 | 38.3 |
| HG995288.1 | 2 | 23.96 | 38.3 |
| HG995289.1 | 3 | 23.81 | 37.9 |
| HG995290.1 | 4 | 23.58 | 38.3 |
| HG995291.1 | 5 | 23.51 | 37.9 |
| HG995292.1 | 6 | 23.29 | 38.4 |
| HG995293.1 | 7 | 22.98 | 38.1 |
| HG995294.1 | 8 | 22.89 | 38.1 |
| HG995295.1 | 9 | 22.53 | 38.3 |
| HG995296.1 | 10 | 22.25 | 38.3 |
| HG995297.1 | 11 | 22.08 | 37.9 |
| HG995298.1 | 12 | 22 | 38 |
| HG995299.1 | 13 | 21.9 | 38.3 |
| HG995300.1 | 14 | 21.03 | 38.2 |
| HG995301.1 | 15 | 20.96 | 38 |
| HG995302.1 | 16 | 20.95 | 38.1 |
| HG995303.1 | 17 | 20.21 | 38.3 |
| HG995304.1 | 18 | 20.07 | 38.2 |
| HG995305.1 | 19 | 19.76 | 38.4 |
| HG995307.1 | 20 | 18.7 | 38.6 |
| HG995308.1 | 21 | 18.6 | 38.4 |
| HG995309.1 | 22 | 18.3 | 37.9 |
| HG995310.1 | 23 | 18.19 | 38.1 |
| HG995311.1 | 24 | 16.97 | 38.4 |
| HG995312.1 | 25 | 15.32 | 38.3 |
| HG995313.1 | 26 | 14.75 | 38.7 |
| HG995314.1 | 27 | 12.11 | 38.9 |
| HG995315.1 | 28 | 11.61 | 38.9 |
| HG995316.1 | 29 | 11.45 | 40.5 |
| HG995317.1 | 30 | 10.61 | 39.3 |
| HG995306.1 | W | 19.6 | 42.6 |
| HG995286.1 | Z | 30.38 | 37.9 |
| HG995318.2 | MT | 0.02 | 18.6 |
| - | unplaced | 0.39 | 48.1 |
Genome annotation report
The H. dysodea genome assembly (GCA_905332915.2) was annotated using the Ensembl rapid annotation pipeline ( Table 1; https://rapid.ensembl.org/Hecatera_dysodea_GCA_905332915.2/). The resulting annotation includes 21,454 transcribed mRNAs from 12,213 protein-coding and 1,652 non-coding genes.
Methods
Sample acquisition and nucleic acid extraction
A female Hecatera dysodea (ilHecDyso1) was collected from Tonbridge, Kent (latitude 51.186305, longitude 0.286464) on 23 June 2020. The specimen was taken from a garden by Gavin Broad (Natural History Museum) using a light trap. The specimen was identified by Gavin Broad and preserved on dry ice.
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI). The ilHecDyso1 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing. Thorax tissue was disrupted using a Nippi Powermasher fitted with a BioMasher pestle. High molecular weight (HMW) DNA was extracted using the Qiagen MagAttract HMW DNA extraction kit. Low molecular weight DNA was removed from a 20 ng aliquot of extracted DNA using 0.8X AMpure XP purification kit prior to 10X Chromium sequencing; a minimum of 50 ng DNA was submitted for 10X sequencing. HMW DNA was sheared into an average fragment size of 12–20 kb in a Megaruptor 3 system with speed setting 30. Sheared DNA was purified by solid-phase reversible immobilisation using AMPure PB beads with a 1.8X ratio of beads to sample to remove the shorter fragments and concentrate the DNA sample. The concentration of the sheared and purified DNA was assessed using a Nanodrop spectrophotometer and Qubit Fluorometer and Qubit dsDNA High Sensitivity Assay kit. Fragment size distribution was evaluated by running the sample on the FemtoPulse system.
RNA was extracted from head tissue of (ilHecDyso1) 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 HiSeq X Ten (10X) instruments. Hi-C data were also generated from abdomen tissue of ilHecDyso1 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 Long Ranger 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 ( Harry, 2022). The mitochondrial genome was assembled using MitoHiFi ( Uliano-Silva et al., 2022), 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 and versions used.
| Software tool | Version | Source |
|---|---|---|
| BlobToolKit | 3.5.2 | Challis et al., 2020 |
| freebayes | 1.3.1-17-gaa2ace8 | Garrison & Marth, 2012 |
| gEVAL | N/A | Chow et al., 2016 |
| Hifiasm | 0.12 | Cheng et al., 2021 |
| HiGlass | 1.11.6 | Kerpedjiev et al., 2018 |
| Long Ranger ALIGN | 2.2.2 | https://support.10xgenomics.com/genome-exome/software/pipelines/latest/advanced/other-pipelines |
| MitoHiFi | 1 | Uliano-Silva et al., 2022 |
| PretextView | 0.2 | Harry, 2022 |
| purge_dups | 1.2.3 | Guan et al., 2020 |
| SALSA | 2.2 | Ghurye et al., 2019 |
Genome annotation
The Ensembl gene annotation system ( Aken et al., 2016) was used to generate annotation for the H. dysodea assembly (GCA_905332915.2). 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).
Ethics and 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. All efforts are undertaken to minimise the suffering of animals used for sequencing. 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: 3 approved]
Data availability
European Nucleotide Archive: Hecatera dysodea (small ranunculus). Accession number PRJEB43532; https://identifiers.org/ena.embl/PRJEB43532. ( Wellcome Sanger Institute, 2021)
The genome sequence is released openly for reuse. The Hecatera dysodea 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.
Author information
Members of the Natural History Museum Genome Acquisition Lab are listed here: https://doi.org/10.5281/zenodo.4790043.
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.
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