Version Changes
Revised. Amendments from Version 1
The sex of the sequences specimen is male, and this has been corrected in the methods section. We have added more information about the assembly method and about the quantities of sequence data generated.
Abstract
We present a genome assembly from an individual male Aporia crataegi (the black-veined white; Arthropoda; Insecta; Lepidoptera; Pieridae). The genome sequence is 230 megabases in span. The complete assembly is scaffolded into 26 chromosomal pseudomolecules, with the Z sex chromosome assembled. Gene annotation of this assembly on Ensembl has identified 10,860 protein coding genes.
Keywords: Aporia crataegi, black-veined white, genome sequence, chromosomal, Lepidoptera
Species taxonomy
Eukaryota; Metazoa; Ecdysozoa; Arthropoda; Hexapoda; Insecta; Pterygota; Neoptera; Endopterygota; Lepidoptera; Glossata; Ditrysia; Papilionoidea; Pieridae; Pierinae; Aporia; Aporia crataegi (Linnaeus, 1758) (NCBI:txid111923).
Background
The black-veined white ( Aporia crataegi) is a large butterfly with distinctive venation on its wings. This species is oligophagous with a larval host plant preference for Prunus and Crataegus spp. and is often considered a pest species in orchards ( Jugovic et al., 2017; Manley, 2008). It is found in a wide variety of habitats including dry grassland, woodland edges, and shrubland ( Tolman & Lewington, 2008). Aporia crataegi is found across the Palaearctic, with populations present in north-west Africa, as well as across Europe and Asia.
The butterfly disappeared from Britain and Ireland around 1925, and the last British specimens were collected from Herne Bay in Kent during the 1920s ( Todisco et al., 2020). It is not understood why the species disappeared from Britain and Ireland; however, climate variability along with other concurrent detrimental conditions, such as parasites, disease, or predation have been suggested as potential reasons ( Pratt, 1983). Several reintroductions have been attempted, but all have been unsuccessful ( Asher et al., 2001), including one purportedly by Winston Churchill after the end of World War II. Given the butterfly's wide Palaearctic distribution, it remains listed as a species of least concern, but more recently it has been reported as extinct in the Czech Republic, the Netherlands ( Van Swaay et al., 2010), and likely South Korea ( Kim et al., 2015). Additionally, abundance and/or range is declining in Austria, Luxembourg, Romania, Ukraine, Albania, France, Latvia, Norway and Serbia ( Van Swaay et al., 2010). No clear consensus exists on the reasons for these declines. We expect that the assembly reported here will facilitate conservation genomic approaches, shedding light on this species' current status ( Todisco et al., 2020). In particular, it will be a valuable resource for any future reintroductions, monitoring, and other local conservation efforts.
Genome sequence report
The genome was sequenced from a single male A. crataegi ( Figure 1) collected from Planoles Station, Catalunya, Spain (latitude 42.3136, longitude 2.0996). The genome was sequenced using Pacific Biosciences single-molecule circular consensus (HiFi) long reads, generating a total of 24.82 Gb (gigabases) from 2.30 million reads, providing an estimated 101-fold coverage. 10X Genomics read clouds were generated for polishing the genome, at approximately 147-fold coverage (72.47 gigabases from 480.1 million reads). Primary assembly contigs were scaffolded with chromosome conformation Hi-C data, which produced 111.79 Gb from 740.32 million reads.
Figure 1. Fore and hind wings of the Aporia crataegi specimens used for sequencing.
Dorsal ( A) and ventral ( B) surface view of wings from specimen PS_AC_246 (ilApoCrat1) from Planoles, Spain, used to generate Pacific Biosciences and 10X genomics data. Dorsal ( C) and ventral ( D) surface view of wings from specimen NU_AC_677 (ilApoCrat2) from Nueno, Spain, used to generate RNA-Seq data.
RNA data was also produced from a different sample, ilApoCrat2, to be used for annotation of the genome: 6.1 Gb were produced from 40.39 million reads.
Manual assembly curation corrected 4 missing/misjoins and removed 5 haplotypic duplications, reducing the assembly length by 0.37% and the scaffold number by 7.14%. The final assembly has a total length of 230 Mb in 26 sequence scaffolds with a scaffold N50 of 25.5 Mb ( Table 1). The complete assembly sequence was assigned to 26 chromosomal-level scaffolds, representing 25 autosomes (numbered by sequence length), and the Z sex chromosome ( Figure 2– Figure 5; Table 2). The assembly has a BUSCO v5.1.2 ( Manni et al., 2021) completeness of 98.5% (single 97.8%, duplicated 0.6%) using the lepidoptera_odb10 reference set ( n=5,286). 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 Aporia crataegi, ilApoCrat1.1: 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 229,694,036 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 (12,847,094 bp, shown in red). Orange and pale-orange arcs show the N50 and N90 chromosome lengths (9,626,953 and 8,364,946 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/ilApoCrat1.1/dataset/ilApoCrat1_1/snail.
Figure 5. Genome assembly of Aporia crataegi, ilApoCrat1.1: Hi-C contact map.
Hi-C contact map of the ilApoCrat1.1 assembly, visualised in HiGlass. Chromosomes are shown in size order from left to right and top to bottom.
Table 1. Genome data for Aporia crataegi, ilApoCrat1.1.
| Project accession data | |
|---|---|
| Assembly identifier | ilApoCrat1.1 |
| Species | Aporia crataegi |
| Specimen | ilApoCrat1 (genome assembly); ilApoCrat2 (RNA-Seq) |
| NCBI taxonomy ID | NCBI:txid129397 |
| BioProject | PRJEB45674 |
| BioSample ID | SAMEA7523355 |
| Isolate information | Male, whole organism (ilApoCrat1); male, thorax (ilApoCrat2) |
| Raw data accessions | |
| PacificBiosciences SEQUEL II | ERR6544652 (2.3 million reads, 24.82 gigabases) |
| 10X Genomics Illumina | ERR6363316-ERR6363319 (480 million reads, 72.47 gigabases) |
| Hi-C Illumina | ERR6363321 (740.32 million reads, 111.79 gigabases) |
| Illumina polyA RNA-Seq | ERR6363320 (40.39 million reads, 6.1 gigabases) |
| Genome assembly | |
| Assembly accession | GCA_912999735.1 |
| Accession of alternate haplotype | GCA_912999795.1 |
| Span (Mb) | 230 |
| Number of contigs | 28 |
| Contig N50 length (Mb) | 9.6 |
| Number of scaffolds | 26 |
| Scaffold N50 length (Mb) | 9.6 |
| Longest scaffold (Mb) | 12.8 |
| BUSCO * genome score | C:98.5%[S:97.8%,D:0.6%],F:0.3%,M:1.2%,n:5286 |
| Genome annotation | |
| Number of protein-coding genes | 10,860 |
| Average length of coding sequence (bp) | 1597.20 |
| Average number of exons per transcript | 8.23 |
| Average exon size (bp) | 259.64 |
| Average intron size (bp) | 1337.70 |
*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/ilApoCrat1.1/dataset/ilApoCrat1_1/busco.
Figure 3. Genome assembly of Aporia crataegi, ilApoCrat1.1.
BlobToolKit GC-coverage plot showing sequence coverage (vertical axis) and GC content (horizontal axis). 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/ilApoCrat1.1/dataset/ilApoCrat1_1/blob.
Figure 4. Genome assembly of Aporia crataegi, ilApoCrat1.1: 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/ilApoCrat1.1/dataset/ilApoCrat1_1/cumulative.
Table 2. Chromosomal pseudomolecules in the genome assembly of Aporia crataegi, ilApoCrat1.1.
| INSDC accession | Chromosome | Size (Mb) | GC% |
|---|---|---|---|
| OU538729.1 | 1 | 12.85 | 32.4 |
| OU538730.1 | 2 | 11.89 | 32.4 |
| OU538731.1 | 3 | 11.06 | 31.9 |
| OU538733.1 | 4 | 10.56 | 32.3 |
| OU538734.1 | 5 | 10.37 | 32.0 |
| OU538735.1 | 6 | 10.35 | 32.1 |
| OU538736.1 | 7 | 9.98 | 32.5 |
| OU538737.1 | 8 | 9.76 | 32.4 |
| OU538738.1 | 9 | 9.64 | 32.2 |
| OU538739.1 | 10 | 9.63 | 32.5 |
| OU538740.1 | 11 | 9.51 | 32.1 |
| OU538741.1 | 12 | 9.47 | 32.2 |
| OU538742.1 | 13 | 9.42 | 32.1 |
| OU538743.1 | 14 | 9.39 | 32.6 |
| OU538744.1 | 15 | 9.37 | 32.4 |
| OU538745.1 | 16 | 8.83 | 32.5 |
| OU538746.1 | 17 | 8.79 | 32.4 |
| OU538747.1 | 18 | 8.74 | 32.1 |
| OU538748.1 | 19 | 8.73 | 32.5 |
| OU538749.1 | 20 | 8.36 | 32.3 |
| OU538750.1 | 21 | 6.60 | 33.2 |
| OU538751.1 | 22 | 4.59 | 33.4 |
| OU538752.1 | 23 | 4.58 | 34.1 |
| OU538753.1 | 24 | 3.40 | 33.5 |
| OU538754.1 | 25 | 3.03 | 33.8 |
| OU538732.1 | Z | 10.79 | 32.1 |
| OU538755.1 | MT | 0.02 | 18.7 |
Genome annotation report
The ilApoCrat1.1 genome has been annotated using the Ensembl rapid annotation pipeline ( Table 1; https://rapid.ensembl.org/Aporia_crataegi_GCA_912999735.1/). The resulting annotation includes 17,867 transcribed mRNAs from 10,860 protein-coding and 1,089 non-coding genes. There are 1.54 coding transcripts per gene and 8.23 exons per transcript. The average transcript length is 11,205.05 bp.
Methods
Sample acquisition and nucleic acid extraction
A male A. crataegi specimen (ilApoCrat1, genome assembly) was collected from Planoles Station, Catalunya, Spain (latitude 42.3136, longitude 2.0996) using a net by Konrad Lohse and Alex Hayward. A second male A. crataegi specimen (ilApoCrat2, RNA-Seq) was collected by Sam Ebdon and Alexander Mackintosh from Nueno, Aragon, Spain (latitude 42.27, longitude –0.45), using a net. Both specimens were formally identified by Konrad Lohse. The samples were snap-frozen at –80°C. Permissions for field sampling were obtained from the Gobierno de Aragon (INAGA/500201/24/2018/0614 to Karl Wotton) and the Generalitat de Catalunya (SF/639).
DNA was extracted from the whole organism of ilApoCrat1 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. RNA (from the thorax of ilApoCrat2) was extracted 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 RNA 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 X (10X) and Illumina HiSeq 4000 (RNA-Seq) instruments. Hi-C data were also generated from remaining whole organism tissue of ilApoCrat1 using the Arima v2 Hi-C kit and sequenced on an Illumina NovaSeq 6000 instrument.
Genome assembly
The Hi-Fi reads were assembled using Hifiasm with the --primary option ( Cheng et al., 2021). Haplotypic duplication was identified and removed with purge_dups ( Guan et al., 2020). One round of polishing of the purged assembly was performed by mapping 10X Genomics read data to the full 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 as described previously ( Howe et al., 2021). Manual curation ( Howe et al., 2021) was performed using 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 |
|---|---|---|
| Hifiasm | 0.12-r304 | Cheng et al., 2021 |
| 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 | 2 | Uliano-Silva et al., 2021 |
| HiGlass | 1.11.6 | Kerpedjiev et al., 2018 |
| PretextView | 0.2.x |
https://github.com/wtsi-hpag/
PretextView |
| BlobToolKit | 2.6.4 | Challis et al., 2020 |
Data availability
European Nucleotide Archive: Aporia crataegi (black veined white). Accession number PRJEB45674; https://identifiers.org/ena.embl/PRJEB45674.
The genome sequence is released openly for reuse. The A. crataegi 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.
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). Fieldwork was supported by a NERC fellowship (NE/L011522/1) and an ERC grant (ModelGenom Land 757648) to KL. AH is supported by a Biotechnology and Biological Sciences Research Council (BBSRC) David Phillips Fellowship (BB/N020146/1).
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
[version 2; peer review: 2 approved, 1 not approved]
Author information
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.5744840.
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.5743293.
Members of the Darwin Tree of Life Consortium are listed here: https://doi.org/10.5281/zenodo.5638618.
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