The genome sequence of the Winter Shade, Tortricodes alternella [Denis & Schiffermüller], 1775

TL;DR

This paper presents the genome sequence of the Winter Shade moth, including its chromosomal structure and gene annotations.

Contribution

The study provides the first genome assembly for Tortricodes alternella, including scaffolded chromosomes and mitochondrial DNA.

Findings

The genome assembly spans 441.2 megabases and includes 22 chromosomal pseudomolecules.

The mitochondrial genome is 15.73 kilobases long and fully assembled.

Gene annotation identified 17,280 protein-coding genes using Ensembl.

Abstract

We present a genome assembly from an individual male
Tortricodes alternella (the Winter Shade; Arthropoda; None; Lepidoptera; Tortricidae). The genome sequence is 441.2 megabases in span. Most of the assembly is scaffolded into 22 chromosomal pseudomolecules, including the Z sex chromosome. The mitochondrial genome has also been assembled and is 15.73 kilobases in length. Gene annotation of this assembly on Ensembl identified 17,280 protein coding genes.

Figures

Photograph of the Tortricodes alternella (ilTorAlte1) specimen used for genome sequencing.

Genome assembly of Tortricodes alternella, ilTorAlte1.1: metrics.The BlobToolKit snail plot 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 441,253,704 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 (39,282,859 bp, shown in red). Orange and pale-orange arcs show the N50 and N90 scaffold lengths (18,827,800 and 13,942,425 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/CANUEM01/dataset/CANUEM01/snail.

Genome assembly of Tortricodes alternella, ilTorAlte1.1: BlobToolKit GC-coverage plot.Sequences are coloured by phylum. Circles are sized in proportion to sequence length. Histograms show the distribution of sequence length sum along each axis. An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/CANUEM01/dataset/CANUEM01/blob.

Genome assembly of Tortricodes alternella, ilTorAlte1.1: BlobToolKit cumulative sequence plot.The grey line shows cumulative length for all sequences. Coloured lines show cumulative lengths of sequences assigned to each phylum using the buscogenes taxrule. An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/CANUEM01/dataset/CANUEM01/cumulative.

Genome assembly of Tortricodes alternella, ilTorAlte1.1: Hi-C contact map of the ilTorAlte1.1 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=O018ksByT3uuMeswRXz6jQ.

Tables

Table 1.. Genome data for Tortricodes alternella, ilTorAlte1.1.

Project accession data
Assembly identifier	ilTorAlte1.1
Species	Tortricodes alternella
Specimen	ilTorAlte1
NCBI taxonomy ID	116138
BioProject	PRJEB57278
BioSample ID	SAMEA110451569
Isolate information	ilTorAlte1, male: whole organism (DNA and Hi-C sequencing) ilTorAlte2: whole organism (RNA sequencing)
Assembly metrics *		Benchmark
Consensus quality (QV)	65.2	≥ 50
k-mer completeness	100.0%	≥ 95%
BUSCO **	C:98.3%[S:97.8%,D:0.5%],F:0.3%,M:1.4%,n:5,286	C ≥ 95%
Percentage of assembly mapped to chromosomes	99.98%	≥ 95%
Sex chromosomes	Z	localised homologous pairs
Organelles	Mitochondrial genome: 15.73 kb	complete single alleles
Raw data accessions
PacificBiosciences SEQUEL II	ERR10462079
Hi-C Illumina	ERR10466813
PolyA RNA-Seq Illumina	ERR12245531
Genome assembly
Assembly accession	GCA_947859335.1
Accession of alternate haplotype	GCA_947858915.1
Span (Mb)	441.2
Number of contigs	87
Contig N50 length (Mb)	10.0
Number of scaffolds	25
Scaffold N50 length (Mb)	18.8
Longest scaffold (Mb)	39.28
Genome annotation
Number of protein-coding genes	17,280
Number of gene transcripts	17,461

Table 2.. Chromosomal pseudomolecules in the genome assembly of Tortricodes alternella, ilTorAlte1.

INSDC accession	Chromosome	Length (Mb)	GC%
OX401968.1	1	39.28	38.0
OX401969.1	2	33.11	38.0
OX401971.1	3	30.74	38.0
OX401972.1	4	25.15	38.5
OX401973.1	5	24.16	38.0
OX401974.1	6	19.27	38.0
OX401975.1	7	18.83	38.5
OX401976.1	8	18.31	38.0
OX401977.1	9	18.11	37.5
OX401978.1	10	17.85	37.5
OX401979.1	11	17.39	38.0
OX401980.1	12	16.7	37.5
OX401981.1	13	16.63	38.0
OX401982.1	14	16.16	38.0
OX401983.1	15	16.06	38.0
OX401984.1	16	15.02	38.0
OX401985.1	17	14.27	38.5
OX401986.1	18	13.94	38.5
OX401987.1	19	13.65	38.0
OX401988.1	20	12.48	38.0
OX401989.1	21	11.99	38.0
OX401970.1	Z	32.06	38.0
OX401990.1	MT	0.02	18.0

Table 3.. Software tools: versions and sources.

Software tool	Version	Source
BlobToolKit	4.2.1	https://github.com/blobtoolkit/blobtoolkit
BUSCO	5.3.2	https://gitlab.com/ezlab/busco
Hifiasm	0.16.1-r375	https://github.com/chhylp123/hifiasm
HiGlass	1.11.6	https://github.com/higlass/higlass
Merqury	MerquryFK	https://github.com/thegenemyers/MERQURY.FK
MitoHiFi	2	https://github.com/marcelauliano/MitoHiFi
PretextView	0.2	https://github.com/wtsi-hpag/PretextView
purge_dups	1.2.3	https://github.com/dfguan/purge_dups
sanger-tol/genomenote	v1.0	https://github.com/sanger-tol/genomenote
sanger-tol/readmapping	1.1.0	https://github.com/sanger-tol/readmapping/tree/1.1.0
YaHS	1.1a.2	https://github.com/c-zhou/yahs

Full text

Species taxonomy

Eukaryota; Opisthokonta; Metazoa; Eumetazoa; Bilateria; Protostomia; Ecdysozoa; Panarthropoda; Arthropoda; Mandibulata; Pancrustacea; Hexapoda; Insecta; Dicondylia; Pterygota; Neoptera; Endopterygota; Amphiesmenoptera; Lepidoptera; Glossata; Neolepidoptera; Heteroneura; Ditrysia; Apoditrysia; Tortricoidea; Tortricidae; Cnephasiinae; Tortricodes; Tortricodes alternella [Denis & Schiffermüller], 1775 (NCBI:txid116138).

Background

Tortricodes alternella, the Winter Shade or Spring Harbinger, is a moth of the Tortricidae family ( Sterling et al., 2023). It has a wingspan of 19–23mm and narrow forewings for a tortrix which are a variably marked in shades of grey/yellow brown with a darker cross-band, whilst the hindwings are pale brown with darker veins. Females are darker with less distinct markings ( Sterling et al., 2023). It is one of the earliest tortricids on the wing, between late January and April, and will come to light ( Sterling et al., 2023). Larvae of T. alternella are up to 15mm long, with a brown head and reddish-brown dorsal surface of the abdomen with white pinacula and dorsal and subdorsal lines, whilst the ventral surface is pale yellow. They feed on various deciduous trees in May and June but predominantly on oaks ( Quercus spp.) and hornbeams ( Carpinus spp.), where they feed within leaves they have spun together ( Kimber, 2024).

T. alternella is classified as common in Britain ( Davis, 2012). It is found in woodland habitats, and sometimes scrub or gardens, throughout England and Wales but is considered local in Scotland and Ireland ( Sterling et al., 2023). Globally, the species is distributed across Europe, between southern Scandinavia and the Mediterranean over to the Balkan Peninsula, with scattered records into western Asia ( GBIF Secretariat, 2024).

The genome of the winter shade, Tortricodes alternella, was sequenced as part of the Darwin Tree of Life Project, a collaborative effort to sequence all named eukaryotic species in the Atlantic Archipelago of Britain and Ireland. Here we present a chromosomal-level genome sequence for Tortricodes alternella, based on a male specimen from Wytham Woods, Oxfordshire.

Genome sequence report

The genome was sequenced from a male Tortricodes alternella ( Figure 1) collected from Wytham Woods, Oxfordshire, UK (51.77, –1.34). A total of 58-fold coverage in Pacific Biosciences single-molecule HiFi long reads was generated. Primary assembly contigs were scaffolded with chromosome conformation Hi-C data. Manual assembly curation corrected 8 missing joins or mis-joins and removed 4 haplotypic duplications, reducing the scaffold number by 3.70%.

Photograph of the Tortricodes alternella (ilTorAlte1) specimen used for genome sequencing.

The final assembly has a total length of 441.2 Mb in 25 sequence scaffolds with a scaffold N50 of 18.8 Mb ( Table 1). The snail plot in Figure 2 provides a summary of the assembly statistics, while the distribution of assembly scaffolds on GC proportion and coverage is shown in Figure 3. The cumulative assembly plot in Figure 4 shows curves for subsets of scaffolds assigned to different phyla. Most (99.98%) of the assembly sequence was assigned to 22 chromosomal-level scaffolds, representing 21 autosomes and the Z sex chromosome. Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size ( Figure 5; Table 2). The Z chromosome was identified based on synteny with Eudemis profundana (GCA_947034925.1) ( Boyes et al., 2023). While not fully phased, the assembly deposited is of one haplotype. Contigs corresponding to the second haplotype have also been deposited. The mitochondrial genome was also assembled and can be found as a contig within the multifasta file of the genome submission.

Table 1.: Genome data for Tortricodes alternella, ilTorAlte1.1.

Genome assembly of Tortricodes alternella, ilTorAlte1.1: metrics.The BlobToolKit snail plot 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 441,253,704 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 (39,282,859 bp, shown in red). Orange and pale-orange arcs show the N50 and N90 scaffold lengths (18,827,800 and 13,942,425 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/CANUEM01/dataset/CANUEM01/snail.

Genome assembly of Tortricodes alternella, ilTorAlte1.1: BlobToolKit GC-coverage plot.Sequences are coloured by phylum. Circles are sized in proportion to sequence length. Histograms show the distribution of sequence length sum along each axis. An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/CANUEM01/dataset/CANUEM01/blob.

Genome assembly of Tortricodes alternella, ilTorAlte1.1: BlobToolKit cumulative sequence plot.The grey line shows cumulative length for all sequences. Coloured lines show cumulative lengths of sequences assigned to each phylum using the buscogenes taxrule. An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/CANUEM01/dataset/CANUEM01/cumulative.

Genome assembly of Tortricodes alternella, ilTorAlte1.1: Hi-C contact map of the ilTorAlte1.1 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=O018ksByT3uuMeswRXz6jQ.

Table 2.: Chromosomal pseudomolecules in the genome assembly of Tortricodes alternella, ilTorAlte1.

The estimated Quality Value (QV) of the final assembly is 65.2 with k-mer completeness of 100.0%, and the assembly has a BUSCO v5.3.2 completeness of 98.3% (single = 97.8%, duplicated = 0.5%), using the lepidoptera_odb10 reference set ( n = 5,286).

Metadata for specimens, barcode results, spectra estimates, sequencing runs, contaminants and pre-curation assembly statistics are given at https://links.tol.sanger.ac.uk/species/116138.

Genome annotation report

The Tortricodes alternella genome assembly (GCA_947859335.1) was annotated at the European Bioinformatics Institute (EBI) on Ensembl Rapid Release. The resulting annotation includes 17,461 transcribed mRNAs from 17,280 protein-coding genes ( Table 1; https://rapid.ensembl.org/Tortricodes_alternella_GCA_947859335.1/Info/Index).

Methods

Sample acquisition and nucleic acid extraction

Specimens of Tortricodes alternella were collected from Wytham Woods, Oxfordshire (biological vice-country Berkshire), UK (latitude 51.77, longitude –1.34) on 2022-03-07 using a light trap. The specimens were collected and identified by Liam Crowley (University of Oxford) and preserved on dry ice. The specimen with ID Ox002042, (ToLID ilTorAlte1) was used for DNA and Hi-C sequencing, and the specimen ID Ox002043 (ToLID ilTorAlte2) was used for RNA sequencing.

The workflow for high molecular weight (HMW) DNA extraction at the Wellcome Sanger Institute (WSI) includes a sequence of core procedures: sample preparation; sample homogenisation, DNA extraction, fragmentation, and clean-up. The sample was prepared for DNA extraction at the WSI Tree of Life Core Laboratory: the ilTorAlte1 sample was weighed and dissected on dry ice ( Jay et al., 2023) and tissue from the whole organism was homogenised using a PowerMasher II tissue disruptor ( Denton et al., 2023a).

HMW DNA was extracted at the WSI Scientific Operations core using the Automated MagAttract v2 protocol ( Oatley et al., 2023). The DNA was sheared into an average fragment size of 12–20 kb in a Megaruptor 3 system with speed setting 31 ( Bates et al., 2023). Sheared DNA was purified by solid-phase reversible immobilisation ( Strickland et al., 2023): in brief, the method employs a 1.8X ratio of AMPure PB beads to sample to eliminate shorter fragments and concentrate the DNA. 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 ilTorAlte2 in the Tree of Life Laboratory at the WSI using the RNA Extraction: Automated MagMax™ mirVana protocol ( do Amaral et al., 2023). The RNA concentration was assessed using a Nanodrop spectrophotometer and a Qubit Fluorometer using the Qubit RNA Broad-Range Assay kit. Analysis of the integrity of the RNA was done using the Agilent RNA 6000 Pico Kit and Eukaryotic Total RNA assay.

Protocols developed by the WSI Tree of Life laboratory are publicly available on protocols.io ( Denton et al., 2023b).

Sequencing

Pacific Biosciences HiFi circular consensus 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) and Illumina NovaSeq 6000 (RNA-Seq) instruments. Hi-C data were also generated from remaining tissue of ilTorAlte1 using the Arima2 kit and sequenced on the Illumina NovaSeq 6000 instrument.

Genome assembly, curation and evaluation

Assembly was carried out with Hifiasm ( Cheng et al., 2021) and haplotypic duplication was identified and removed with purge_dups ( Guan et al., 2020). The assembly was then scaffolded with Hi-C data ( Rao et al., 2014) using YaHS ( Zhou et al., 2023). The assembly was checked for contamination and corrected as described previously ( Howe et al., 2021). Manual curation was performed using HiGlass ( Kerpedjiev et al., 2018) and PretextView ( Harry, 2022). The mitochondrial genome was assembled using MitoHiFi ( Uliano-Silva et al., 2023), which runs MitoFinder ( Allio et al., 2020) or MITOS ( Bernt et al., 2013) and uses these annotations to select the final mitochondrial contig and to ensure the general quality of the sequence.

A Hi-C map for the final assembly was produced using bwa-mem2 ( Vasimuddin et al., 2019) in the Cooler file format ( Abdennur & Mirny, 2020). To assess the assembly metrics, the k-mer completeness and QV consensus quality values were calculated in Merqury ( Rhie et al., 2020). This work was done using Nextflow ( Di Tommaso et al., 2017) DSL2 pipelines “sanger-tol/readmapping” ( Surana et al., 2023a) and “sanger-tol/genomenote” ( Surana et al., 2023b). The genome was analysed within the BlobToolKit environment ( Challis et al., 2020) and BUSCO scores ( Manni et al., 2021; Simão et al., 2015) were calculated.

Table 3 contains a list of relevant software tool versions and sources.

Genome annotation

The BRAKER2 pipeline ( Brůna et al., 2021) was used in the default protein mode to generate annotation for the Tortricodes alternella assembly (GCA_947859335.1) in Ensembl Rapid Release at the EBI.

Wellcome Sanger Institute – Legal and Governance

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’, which can be found in full on the Darwin Tree of Life website here. 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.

Further, 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 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.