The genome sequence of the clouded yellow, Colias crocea (Geoffroy, 1785).

We present a genome assembly from an individual female Colias crocea (also known as Colias croceus; the clouded yellow; Arthropoda; Insecta; Lepidoptera; Pieridae). The genome sequence is 325 megabases in span. The complete assembly is scaffolded into 32 chromosomal pseudomolecules, with the W and Z sex chromosome assembled. Gene annotation of this assembly on Ensembl has identified 13,803 protein coding genes. Copyright:

Species taxonomy

Eukaryota; Metazoa; Ecdysozoa; Arthropoda; Hexapoda; Insecta; Pterygota; Neoptera; Endopterygota; Lepidoptera; Glossata; Ditrysia; Papilionoidea; Pieridae; Coliadinae; Colias; Colias crocea (also known as Colias croceus) (Geoffroy, 1785) (NCBI:txid72248).

Background

Colias crocea (or croceus), the clouded yellow, is a butterfly found in Europe, the middle east, and north Africa. This continuously-brooded migratory species visits the UK in the end of spring and summer, supplementing a small breeding population in the south. The larvae feed on a wide variety of leguminous plants, such as clovers ( Trifolium sp.), alfalfa ( Medicago sativa) and vetches ( Vicia sp.). Despite recent declines, C. crocea has seen a large increase in both abundance and occurrence in the last 50 years in the British Isles ( Fox ) and is listed as Least Concern in the IUCN Red List (Europe) ( van Swaay ). A white polymorphism known as Alba (form helice) is associated with an alternative life-history strategy, where females reallocate wing pigment resources to somatic and reproductive development. This is associated with the insertion of a transposable element downstream of the homeobox transcription factor BarH-1 ( Woronik ). Colias crocea has 31 pairs of chromosomes, a genome size of approximately 318.6 Mb ( Woronik ), and is female heterogametic (WZ). We note the recent production of a high-quality genome assembly for C. crocea ( Woronik ), and believe the sequence described here, generated as part of the Darwin Tree of Life project, will further aid understanding of the biology and ecology of this butterfly.

Genome sequence report

The genome was sequenced from a single female C. crocea collected from Bujaruelo, Aragon, Spain (latitude 42.7, longitude -0.1) ( Figure 1). A total of 68-fold coverage in Pacific Biosciences single-molecule long reads and 91-fold coverage in 10X Genomics read clouds were generated. Primary assembly contigs were scaffolded with chromosome conformation Hi-C data. Manual assembly curation corrected 6 missing/misjoins, reducing the assembly length by 0.8% and the scaffold number by 13.5%.

Figure 1.

Fore and hind wings of Colias crocea specimen from which the genome was sequenced.

( A) Dorsal surface view of wings from specimen BU_CC_715 (ilColCroc2) from Bujaruelo, Spain, used to generate Pacific Biosciences and 10X genomics data. ( B) Ventral surface view of wings from specimen BU_CC_715 (ilColCroc2) from Bujaruelo, Spain, used to generate Pacific Biosciences and 10X genomics data.

The final assembly has a total length of 325 Mb in 33 sequence scaffolds with a scaffold N50 of 11 Mb ( Table 1). Of the assembly sequence, 100% 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 ( Simão ) v5.1.2 completeness of 99.0% (single 98.7%, duplicated 0.3%, fragmented 0.2%, missing 0.8%) 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.

Table 1.

Genome data for Colias crocea, ilColCroc2.1.

Project accession data
Assembly identifier	ilColCroc2.1
Species	Colias crocea (also known as Colias croceus)
Specimen	ilColCroc2
NCBI taxonomy ID	NCBI:txid72248
BioProject	PRJEB42878
BioSample ID	SAMEA7523360
Isolate information	Female, abdomen/thorax
Raw data accessions
PacificBiosciences SEQUEL II	ERR6558184
10X Genomics Illumina	ERR6054394-ERR6054397
Hi-C Illumina	ERR6054398
Illumina PolyA RNAseq	ERR6054399
Genome assembly
Assembly accession	GCA_905220415.1
Accession of alternate haplotype	GCA_905220445.1
Span (Mb)	325
Number of contigs	42
Contig N50 length (Mb)	11
Number of scaffolds	33
Scaffold N50 length (Mb)	11
Longest scaffold (Mb)	15
BUSCO* genome score	C:99.0%[S:98.6%,D:0.4%],F:0.2%,M:0.8%,n:1658
Gene annotation
Number of protein-coding genes	13,830
Average length of protein coding sequence (bp)	1.631
Average number of exons per gene	8
Average exon size (bp)	359
Average intron size (bp)	2,027

Figure 2.

Genome assembly of Colias crocea, ilColCroc2.1: 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 324,912,214 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 (17,237,107 bp, shown in red). Orange and pale-orange arcs show the N50 and N90 chromosome lengths (11,204,669 and 7,474,634 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/ilColCroc2.1/dataset/ilColCroc2_1/snail.

Figure 5.

Genome assembly of Colias crocea, ilColCroc2.1: Hi-C contact map.

Hi-C contact map of the ilColCroc2.1 assembly, visualised in HiGlass.

Table 2.

Chromosomal pseudomolecules in the genome assembly of Colias crocea, ilColCroc2.1.

INSDC accession	Chromosome	Size (Mb)	GC%
HG991959.1	1	15.09	34.1
HG991960.1	2	13.25	33.8
HG991961.1	3	13.17	34
HG991962.1	4	12.85	33.9
HG991963.1	5	12.69	33.4
HG991964.1	6	12.66	33.1
HG991965.1	7	12.04	33.3
HG991966.1	8	11.65	33.2
HG991967.1	9	11.34	33.4
HG991968.1	10	11.32	33.3
HG991969.1	11	11.31	33.4
HG991970.1	12	11.20	33.4
HG991971.1	13	11.13	33.1
HG991972.1	14	10.75	33.3
HG991973.1	15	10.73	33.5
HG991974.1	16	10.69	33.4
HG991975.1	17	10.66	33.6
HG991976.1	18	10.16	33.7
HG991977.1	19	9.83	33.4
HG991978.1	20	9.70	33.3
HG991979.1	21	9.58	33.7
HG991980.1	22	8.79	33.7
HG991981.1	23	8.06	36
HG991982.1	24	7.97	32.9
HG991983.1	25	7.88	32.9
HG991984.1	26	7.47	33.5
HG991985.1	27	7.27	33.3
HG991986.1	28	5.88	33.7
HG991987.1	29	5.28	33.6
HG991988.1	30	5.08	34.5
HG991989.1	W	2.16	36
HG991958.1	Z	17.24	33.9
HG991990.1	MT	0.02	18.7

Genome assembly of Colias crocea, ilColCroc2.1: GC coverage.

BlobToolKit GC-coverage plot. Chromosomes are coloured by phylum. Circles are sized in proportion to scaffold 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/ilColCroc2.1/dataset/ilColCroc2_1/blob.

Genome assembly of Colias crocea, ilColCroc2.1: 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/ilColCroc2.1/dataset/ilColCroc2_1/cumulative.

Gene annotation

The Ensembl gene annotation system ( Aken ) was used to generate annotation for the Colias crocea assembly ( GCA_905220415.1, 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 ). Prediction tools, CPC2 ( Kang ) and RNAsamba ( Camargo ), were used to aid determination of protein coding genes.

Methods

Sample acquisition and nucleic acid extraction

A female (ilColCroc2) and a male (ilColCroc3) C. crocea were collected from Bujaruelo, Aragon, Spain (latitude 42.7, longitude -0.1) by Sam Ebdon, Alex Macintosh (both University of Edinburgh), Alex Hayward and Karl Wotton (both University of Exeter). Samples were collected using a net and snap-frozen in liquid nitrogen.

DNA was extracted at the Wellcome Sanger Institute (WSI) Scientific Operations core from the thorax of ilColCroc2 using the Qiagen MagAttract HMW DNA kit, according to the manufacturer’s instructions. RNA was extracted from the thorax of ilColCroc3 in the Tree of Life Laboratory at the WSI using TRIzol (Invitrogen), 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 sequencing libraries were constructed according to the manufacturers’ instructions. SPoly(A) RNA-Seq libraries were constructed using the NEB Ultra II RNA Library Prep kit. Sequencing was performed by the Scientific Operations core at the Wellcome Sanger Institute on Pacific Biosciences SEQUEL II (HiFi), Illumina HiSeq X (10X) and Illumina HiSeq 4000 (RNA-Seq) instruments. Hi-C data were generated from abdomen tissue of ilColCroc2 using the Arima v2.0 kit and sequenced on HiSeq X.

Genome assembly

Assembly was carried out with Hifiasm ( Cheng ). Haplotypic duplication was identified and removed with purge_dups ( Guan ). 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 ) using SALSA2 ( Ghurye ). The assembly was checked for contamination and corrected using the gEVAL system ( Chow ) as described previously ( Howe ). Manual curation was performed using gEVAL, HiGlass ( Kerpedjiev ) and Pretext. The mitochondrial genome was assembled using MitoHiFi ( Uliano-Silva ). The genome was analysed and BUSCO scores generated within the BlobToolKit environment ( Challis ). 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	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	1.0	https://github.com/marcelauliano/MitoHiFi
gEVAL	N/A	Chow et al., 2016
HiGlass	1.11.6	Kerpedjiev et al., 2018
PretextView	0.1.x	https://github.com/wtsi-hpag/PretextView
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 Wellcome Sanger Institute 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: Colias croceus (clouded yellow). Accession number PRJEB42949; https://identifiers.org/ena.embl/PRJEB42949.

The genome sequence is released openly for reuse. The C. crocea 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 Darwin Tree of Life Barcoding collective are listed here: https://doi.org/10.5281/zenodo.4893704.

Members of the Wellcome Sanger Institute Tree of Life programme collective are listed here: https://doi.org/10.5281/zenodo.5377053.

Members of Wellcome Sanger Institute Scientific Operations: DNA Pipelines collective are listed here: https://doi.org/10.5281/zenodo.4790456.

Members of the Tree of Life Core Informatics collective are listed here: https://doi.org/10.5281/zenodo.5013542.

Members of the Darwin Tree of Life Consortium are listed here: https://doi.org/10.5281/zenodo.4783559.

The authors present a highly contiguous and complete genome assembly for the Pierid butterfly, Colias crocea. The existence of a female-limited color polymorphism, in which females are either white or orange, has made this and other species in the genus emerging model systems for the study of alternative life history strategies and the maintenance of ancient polymorphisms.

For this genome assembly, the authors used a combination of sequence data from an orange female, providing detailed information about the sequencing and assembly steps. The authors also used RNAseq data sampled from the thorax of a single adult male to generate an annotation for this assembly, which may limit its scope for describing both the protein coding and noncoding features of this assembly. Overall, I agree with the previous reviewer that a genome assembly of this quality is highly beneficial (and has likely already been useful) for the study of C. crocea and for comparative research in butterfly genomics.

Critiques:

I support the critiques of the first reviewer: (1) that more details should be provided about the parameters and options for software used to generate both the assembly and the annotation; and (2) that the manuscript would benefit from a more detailed explanation of the differences between this assembly and existing genomic resources.

Details are lacking about the annotation. Given the annotation was made using RNA from a single male thorax, it is possible that there were many important coding and noncoding transcripts missing in the sample. The protein prediction tools subsequently used to improve the annotation may have filled these gaps, but I think it is important that the authors both acknowledge these limitations and include some evaluation of annotation quality. The authors could run BUSCO on the protein set or could compare orthologs between this and other well-annotated Lepidopteran gene sets. Also, summary statistics (e.g., average length of protein coding sequence) provided in Table 1 should include some measure of variation (95% CI, etc.)

Are sufficient details of methods and materials provided to allow replication by others?

Partly

Is the rationale for creating the dataset(s) clearly described?

Yes

Are the datasets clearly presented in a useable and accessible format?

Yes

Are the protocols appropriate and is the work technically sound?

Yes

Reviewer Expertise:

Evolutionary ecology, gene expression, alternative splicing

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Summary of the article and contributions:

The authors generate a high quality genome for the butterfly Colias crocea using PacBio, 10X Genomics, and HiC sequencing data. These sequencing datasets were generated from an orange C. crocea female. The resulting genome was annotated using RNA-Seq data from a male  C. crocea. This is a high quality genome that is more contiguous and complete than a previously published  C. crocea genome (Woronik et al., 2019 ). Because this butterfly species harbors structural variation that results in an alternative life history strategy, such high quality genomes are necessary for in depth study of these (and potentially other) important genomic regions. Overall, this genome will contribute to the understanding of this butterfly’s biology and ecology.

Critiques:

The authors reported the software and versions used for their assembly. However, I could not find a place where the various options used in those pipelines were stated. Would it be possible for the authors to include the options used in the assembly and annotation commands? This would increase the reproducibility of this work. This could be done by adding another column to Table 3 "Options used" or by including their scripts as a supplemental file.

The authors point out that another high quality genome exists for C. crocea. This genome was generated from Alba females and annotated using RNA-Seq data from the wings and abdomens of several female pupas. Adding a sentence to the "Background" that highlights the differences between the datasets could be useful for readers interested in using this data as they complement each other nicely.

Are sufficient details of methods and materials provided to allow replication by others?

Partly

Is the rationale for creating the dataset(s) clearly described?

Yes

Are the datasets clearly presented in a useable and accessible format?

Yes

Are the protocols appropriate and is the work technically sound?

Yes

Reviewer Expertise:

Evolutionary genomics and developmental genetics

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.