The genome sequence of the large white, Pieris brassicae (Linnaeus, 1758)

Abstract

We present a genome assembly from an individual female Pieris brassicae (the large white; Arthropoda; Insecta; Lepidoptera; Pieridae). The genome sequence is 292 megabases in span. The majority of the assembly is scaffolded into 16 chromosomal pseudomolecules, with the W and Z sex chromosome assembled. Gene annotation of this assembly on Ensembl has identified 12,229 protein coding genes.

Species taxonomy

Eukaryota; Metazoa; Ecdysozoa; Arthropoda; Hexapoda; Insecta; Pterygota; Neoptera; Endopterygota; Lepidoptera; Glossata; Ditrysia; Papilionoidea; Pieridae; Pierinae; Pieris; Pieris brassicae (Linnaeus, 1758) (NCBI:txid7116).

Introduction

The large white, Pieris brassicae, is a Palearctic butterfly species that is common in Europe, North Africa, and Asia. P. brassicae larvae typically feed on Brassicaceae species, including cultivated species with agricultural importance such as Brassica oleracea. It has been unintentionally introduced to New Zealand, Chile, and South Africa, although it was later eradicated from New Zealand in 2016 ( Phillips et al., 2020). It overwinters as a pupa and is multivoltine. While P. brassicae has been listed as Least Concern in the IUCN Red List (Europe), the Madeiran large white, P. wollastoni (previously considered a subspecies of P. brassicae), has not been observed since 1986 and is possibly extinct ( IUCN, 2009). P. brassicae has 15 pairs of chromosomes with the female being heterogametic ( Bigger, 1975). This karyotype is unusual , as species in the genus Pieris typically possess between 24 and 28 pairs of chromosomes ( Robinson, 1971). Its genome size has been estimated with flow cytometry at approximately 260 Mb ( Mackintosh et al., 2019).

Genome sequence report

The genome was sequenced from a single female P. brassicae (ilPieBrab1) collected from East Linton, Scotland (latitude 55.977161, longitude -2.667545); Hi-C data were generated from a male P. brassicae (ilPieBrab3) collected from the same location ( Figure 1). A total of 92-fold coverage in Pacific Biosciences single-molecule long reads (N50 14 kb) and 138-fold coverage in 10X Genomics read clouds were generated. Primary assembly contigs were scaffolded with chromosome conformation Hi-C data, which was obtained from a different individual (ilPieBrab3). Manual assembly curation corrected 25 missing joins, reducing the scaffold number by 5.87%, and increasing the scaffold N50 by 1.21%.

Figure 1. Fore and hind wings of Pieris brassicae specimens from which the genome was sequenced.

(A) Dorsal surface view of wings from specimen SC_PB_1356 ( ilPieBrab1) from East Linton, used to generate Pacific Biosciences and 10X genomics data. (B) Ventral surface view of wings from specimen SC_PB_1356 (ilPieBrab1) from East Linton, used to generate Pacific Biosciences and 10X genomics data. (C) Dorsal surface view of wings from specimen SC_PB_1357 (ilPieBrab2) from East Linton, used to generate RNASeq data. (B) Ventral surface view of wings from specimen SC_PB_1357 (ilPieBrab2) from East Linton, used to generate RNASeq data. (A) Dorsal surface view of wings from specimen SC_PB_1358 (ilPieBrab3) from East Linton, used to generate Hi-C data. (B) Ventral surface view of wings from specimen SC_PB_1358 (ilPieBrab3) from East Linton, used to generate Hi-C data.

The final assembly has a total length of 292 Mb in 402 sequence scaffolds with a scaffold N50 of 22 Mb ( Table 1). Of the assembly sequence, 95.46% was assigned to 16 chromosomal-level scaffolds, representing 14 autosomes (numbered by sequence length), and the W and Z sex chromosome ( Figure 2– Figure 5; Table 2). The W chromosome is fragmented as the assembly was scaffolded to an individual of a different sex (ilPieBrab3). The assembly has a BUSCO ( Simão et al., 2015) completeness of 99.0% 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 Pieris brassicae, ilPieBrab1.1: 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 292,341,157 bp assembly. The distribution of scaffold lengths is shown in dark grey with the plot radius scaled to the longest chromosome present in the assembly (23,587,467 bp, shown in red). Orange and pale-orange arcs show the N50 and N90 chromosome lengths (21,549,451 and 6,322,985 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/ilPieBrab1.1/dataset/CAJHUI01/snail.

Figure 5. Genome assembly of Pieris brassicae, ilPieBrab1.1: Hi-C contact map.

Hi-C contact map of the ilPieBrab1.1 assembly, visualised in HiGlass. Chromosomes are shown in order of size from left to right and top to bottom.

BlobToolKit blob and cumulative sequence plots show that the W chromosome has regions with microsporidian origin ( Figure 3, Figure 4). However, these regions in the read sets are short, do not match across the rest of the scaffold and do not contain any contigs with microsporidian ribosomal subunits. This indicates that this feature is unlikely to be contamination and is more likely to be the result of integration of microsporidian sequence into the genome.

Figure 3. Genome assembly of Pieris brassicae, ilPieBrab1.1: 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/ilPieBrab1.1/dataset/CAJHUI01/blob.

Figure 4. Genome assembly of Pieris brassicae, ilPieBrab1.1: 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/ilPieBrab1.1/dataset/CAJHUI01/cumulative.

Table 1. Genome data for Pieris brassicae, ilPieBrab1.1.

Project accession data
Assembly identifier	ilPieBrab1.1
Species	Pieris brassicae
Specimen	ilPieBrab1 (genome assembly), ilPieBrab2 (RNA-Seq), ilPieBrab3 (Hi-C)
NCBI taxonomy ID	NCBI:txid7116
BioProject	PRJEB41887
BioSample ID	SAMEA7532735
Isolate information	Female, whole organism (ilPieBrab1)
	Males, whole organisms (ilPieBrab2, ilPieBrab3)
Raw data accessions
PacificBiosciences SEQUEL II	ERR6594497
10X Genomics Illumina	ERR6002738-ERR6002741
Hi-C Illumina	ERR6002742
Illumina PolyA RNA-Seq	ERR6594497
Genome assembly
Assembly accession	GCA_905147105.1
Accession of alternate haplotype	GCA_905147085.1
Span (Mb)	292
Number of contigs	431
Contig N50 length (Mb)	21
Number of scaffolds	402
Scaffold N50 length (Mb)	22
Longest scaffold (Mb)	24
BUSCO * genome score	C:99.0%[S:98.7%,D:0.3%],F:0.3%, M:0.7%,n:5286
Gene annotation
Number of protein coding genes	12,229
Average coding sequence length (bp)	1,736
Average number of exons per transcript	10
Average exon size (bp)	327
Average intron size (bp)	1687

*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/ilPieBrab1.1/dataset/CAJHUI01/busco.

Table 2. Chromosomal pseudomolecules in the genome assembly of Pieris brassicae, ilPieBrab1.1.

INSDC accession	Chromosome	Size (Mb)	GC%
LR989932.1	1	23.59	33.5
LR989933.1	2	23.11	33.5
LR989934.1	3	23.07	33.7
LR989935.1	4	22.87	33.6
LR989936.1	5	22.82	33.6
LR989937.1	6	21.88	33.5
LR989938.1	7	21.55	33.7
LR989939.1	8	21.29	33.6
LR989940.1	9	19.44	33.7
LR989941.1	10	19.01	33.8
LR989942.1	11	14.79	33.7
LR989944.1	12	11.23	34.1
LR989945.1	13	6.32	35.2
LR989946.1	14	5.84	36
LR989947.1	W	5.29	37
LR989943.1	Z	12.97	33.9
LR989948.1	MT	0.02	20.7
-	Unplaced	17.25	39.1

Gene annotation

The Ensembl gene annotation system ( Aken et al., 2016) was used to generate annotation for the Pieris brassicae assembly (GCA_905147105.1, see https://rapid.ensembl.org/Pieris_brassicae_GCA_905147105.1/; Table 1). The 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) and OrthoDB ( Kriventseva et al., 2008). Prediction tools, CPC2 ( Kang et al., 2017) and RNAsamba ( Camargo et al., 2020), were used to aid determination of protein coding genes.

Methods

Sample acquisition and nucleic acid extraction

A female (ilPieBrab1) and two male (ilPieBrab2, ilPieBrab3) P. brassicae ( Figure 1) were collected from East Linton, Scotland (latitude 55.977161, longitude -2.667545) using a net by Konrad Lohse, University of Edinburgh, who also identified the samples. The samples were snap-frozen in liquid nitrogen from live.

DNA was extracted from the whole organism of ilPieBrab1 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 (also from the whole organism) was extracted from ilPieBrab2 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 generated from the whole organism of ilPieBrab3 using the Qiagen EpiTect Hi-C 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 was performed using gEVAL, HiGlass ( Kerpedjiev et al., 2018) and Pretext. 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	1.0	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
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	2.6.2	Challis et al., 2020

Ethical/compliance issues

The materials that have contributed to this genome note were supplied by a Tree of Life collaborator. The WSI 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 undertaken according to a Research Collaboration Agreement or Material Transfer Agreement entered into by the Tree of Life collaborator, Genome Research Limited (operating as the Wellcome Sanger Institute) and in some circumstances other Tree of Life collaborators.

Data availability

European Nucleotide Archive: Pieris brassicae (large white). Accession number PRJEB42142; https://identifiers.org/ena.embl/PRJEB42142.

The genome sequence is released openly for reuse. The P. brassicae 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). KL is supported by a NERC fellowship (NE/L011522/1) and an ERC grant (ModelGenom Land 757648). AM is supported by a E4 PhD studentship.

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