The genome sequence of the Diamondback Moth, Plutella xylostella (Linnaeus, 1758)

We present a genome assembly from an individual male Plutella xylostella (the Diamondback Moth; Arthropoda; Insecta; Lepidoptera; Plutellidae). The genome sequence is 323.3 megabases in span. Most of the assembly is scaffolded into 31 chromosomal pseudomolecules, including the Z sex chromosome. The mitochondrial genome has also been assembled and is 35.12 kilobases in length. Gene annotation of this assembly on Ensembl identified 17,190 protein coding genes.


Background
The Diamondback Moth, Plutella xylostella, is a micromoth in the Plutellidae family, previously in the family Yponomeutidae.Members of this family are often characterised by elongated forewings with a distinctive shape (Sterling & Parsons, 2018).Although there are only seven species, the Plutellidae family has significant relevance in agriculture, as the Diamondback Moth is a notorious global pest.This moth is common in Britain and Ireland, arriving on the shores in great numbers.The adults fly by day and come to light.There are several broods each year, more in warmer areas (Sterling & Parsons, 2018).
The P. xylostella larva is the main pest of cruciferous crops worldwide (Zalucki et al., 2012).It is the most widely distributed of all lepidopteran pests (Talekar & Shelton, 1993), and indeed has the widest distribution of all Lepidoptera (Furlong et al., 2013).The annual cost of losses of crop production to P. xylostella infestations have been estimated at up to 5 billion USD (Furlong et al., 2013).It has also rapidly evolved field resistance to all major classes of synthetic and biological insecticides (Furlong et al., 2013) through mutations in insecticidal receptors, including Bt toxins (Baxter et al., 2011), and overexpression of detoxification genes.Pesticide resistance might also be conferred by the gut microbiota (Xia et al., 2013;Xia et al., 2018).
To understand the capacity of this moth to respond to environmental stressors and develop resistance to insecticides, it has been the target of many molecular studies.In 2013, two parallel projects reported the first P. xylostella reference genomes, also establishing a genomic and transcriptomic database (Jouraku et al., 2013;You et al., 2013).A chromosome-level haploid genome assembly was generated by a trio binning strategy (Ward et al., 2021).An analysis of 532 genomes through resequencing and variation analysis provided evidence that P. xylostella originated in South America and expanded throughout the world in three major expansions (You et al., 2020).
The genome of P. xylostella was sequenced using the Darwin Tree of Life pipeline, a part of the broader collaborative effort to sequence all named eukaryotic species in the Atlantic Archipelago of Britain and Ireland.This methodological approach represents a standardised procedure ensuring quality and consistency in genomic analysis.Here we present a chromosomally complete genome sequence for P. xylostella, based on one male specimen from Wytham Woods, Oxfordshire, UK.

Genome sequence report
The genome was sequenced from one male Plutella xylostella (Figure 1) collected from Wytham Woods, Oxfordshire, UK (51.77,.A total of 77-fold coverage in Pacific Biosciences single-molecule HiFi long reads and 148-fold coverage in 10X Genomics read clouds were generated.Primary assembly contigs were scaffolded with chromosome conformation Hi-C data.Manual assembly curation corrected 8 missing joins or mis-joins and removed 2 haplotypic duplications, reducing the assembly length by 0.11% and the scaffold number by 5.71%, and increasing the scaffold N50 by 1.97%. The final assembly has a total length of 323.3 Mb in 33 sequence scaffolds with a scaffold N50 of 11.3 Mb (Table 1).Most (99.98%) of the assembly sequence was assigned to 31 chromosomal-level scaffolds, representing 30 autosomes and the Z sex chromosome.Chromosome-scale scaffolds confirmed by the Hi-C data are named according to synteny with P. xylostella genome assembly GCA_019096205.1 (Figure 2-Figure 5; Table 2).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.
Metadata for specimens, spectral estimates, sequencing runs, contaminants and pre-curation assembly statistics can be found at https://links.tol.sanger.ac.uk/species/51655.

Genome annotation report
The Plutella xylostella genome assembly (GCA_932276165.1)was annotated using the Ensembl rapid annotation pipeline

Sample acquisition and nucleic acid extraction
The specimens used in this study were collected from Wytham Woods, Oxfordshire (biological vice-county Berkshire), UK (latitude 51.77, longitude -1.32) on 2019-09-21, using a light trap.Douglas Boyes (University of Oxford) collected and identified the specimens.The specimens were snap-frozen on dry ice.The specimen used for genome sequencing was a male Plutella xylostella (specimen ID Ox000293, ToLID ilPluXylo3) while the specimen used for Hi-C sequencing had specimen ID Ox000294 (ToLID ilPluXylo4).
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI).The ilPluXylo3 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing.Tissue from the whole organism 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 the 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      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.
Table 3 contains a list of relevant software tool versions and sources.

Genome annotation
The Ensembl gene annotation system (Aken et al., 2016) was used to generate annotation for the Plutella xylostella assembly (GCA_932276165.1).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).

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.
Table 3. Software tools: versions and sources.

Software tool Version
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 1.There's a small confusion in the methods, as it is stated in the 2nd paragraph of the methods that tissue from ilPluXylo3 was 'set aside for Hi-C sequencing'.However, in all other parts of the report it is made clear that tissue from a separate individual, ilPluXylo4, was used for Hi-C.
2. In the methods for genome annotation, what transcriptomic data was used?What tissues/life stages were it from, and where can that data be found?This is important for the replicability of the work.
3. Given that a chromosome-level assembly for this species already exists, a brief sentence comparing the two assemblies could be added.Since this paper is just a data note, a large amount of comparative analysis or interpretation would be inappropriate; however, a quick statement noting the similarity in size/structure would be useful for readers.
Is the rationale for creating the dataset(s) clearly described?Yes

Are the protocols appropriate and is the work technically sound? Yes
Are sufficient details of methods and materials provided to allow replication by others?

Partly
Are the datasets clearly presented in a useable and accessible format?Yes Competing Interests: No competing interests were disclosed.
Reviewer Expertise: I work in the field of insect evolutionary genomics.I have been involved with several genome sequencing projects, including that of the lepidopteran pest Helicoverpa zea.
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.

Jerome H L Hui
The Chinese University of Hong Kong, Hong Kong, Hong Kong Boyes and colleagues report the genome sequence of a male diamondblack/cabbage moth Plutella xylostella (Linnaeus, 1758).As one can judge from its common name, it is an important pest of brassicaceous crops such as cabbage.This species is well known for its migratory tendencies and now become a cosmopolitan species.In Britain, the immediate sources of this species have been suggested to be coming from countries in the western part of continental Europe ).Nevertheless, it is necessary to point out that this report contains a much improved high-quality genome resource.It will be useful for a range of further studies, including from not limited to, revealing their population structures, dissecting host plant-insect relationships, to understanding their evolution with other lepidopterans, which have important values in both basic knowledge and applications.
This genome resource is excellent from the summary statistics, with high BUSCO numbers, high sequence continuity (scaffold N50), and majority of sequences contained on the 32 pseudochromosomes (plus mitochondrion).All in all, this is another valuable contribution by the Darwin Tree of Life Consortium.Competing Interests: I have published with Peter Holland more than three years ago, and confirm that this potential conflict of interest did not affect my ability to write an objective and unbiased review of the article.
Reviewer Expertise: Genomics, evolution, invertebrates 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.

Figure 2 .
Figure 2. Genome assembly of Plutella xylostella, ilPluXylo3.1: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 323,337,879 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 (16,174,618 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (11,339,310 and 8,635,333 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/Plutella%20xylostella/dataset/CAKNZZ01/snail.

Figure 3 .
Figure 3. Genome assembly of Plutella xylostella, ilPluXylo3.1: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/Plutella%20xylostella/dataset/CAKNZZ01/blob.

Figure 5 .
Figure 5. Genome assembly of Plutella xylostella, ilPluXylo3.1:Hi-C contact map of the ilPluXylo3.1 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=TaoX1aegQfituIk_tZJ_8Q.

Figure 4 .
Figure 4. Genome assembly of Plutella xylostella, ilPluXylo3.1: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/Plutella%20xylostella/dataset/CAKNZZ01/cumulative.
(Wainwright et al 2020 Insects).As the authors have pointed out, certain level of molecular data of this species have already been obtained previously, including transcriptomes and draft genomes (N50 = 2.2 kb and 737kb from two studies, Jouraku et al 2013 BMC Genomics [Ref 2]; You et al 2013 Nature Genetics [Ref 3] 2. You M, Yue Z, He W, Yang X, et al.: A heterozygous moth genome provides insights into herbivory and detoxification.Nat Genet.2013; 45 (2): 220-5 PubMed Abstract | Publisher Full Text Is the rationale for creating the dataset(s) clearly described?Yes Are the protocols appropriate and is the work technically sound?Yes Are sufficient details of methods and materials provided to allow replication by others?Yes Are the datasets clearly presented in a useable and accessible format?Yes