The genome sequence of common ivy, Hedera helix L., 1753

We present a genome assembly from a specimen of Hedera helix (common ivy; Streptophyta; Magnoliopsida; Apiales; Araliaceae). The genome sequence is 1,199.4 megabases in span. Most of the assembly is scaffolded into 24 chromosomal pseudomolecules. The mitochondrial and plastid genomes have also been assembled and are 609.2 and 162.2 kilobases in length respectively.


Background
The common ivy, Hedera helix L., is a vigorous self-clinging evergreen perennial vine that is one of the most familiar plant species in the British flora.Ivy is common across most of Britain, except parts of northern Scotland.It is much rarer in Ireland, but it is native to much of Europe from southern Scandinavia to Turkey.It forms dense ground cover, particularly in secondary woodland.Here, it acts as a keystone species, with the greenish-yellow flowers providing a rich source of nectar for insects in the autumn and the purplish-black berries being a source of food for birds in the spring (Metcalfe, 2005).The common ivy, as well as other introduced ivy species, are widespread in and around gardens, where they are used as climbers to cover garden structures, particularly in shaded situations.Common ivy has widely escaped from gardens and is now frequently found as a non-native alien around the globe (Biggerstaff & Beck, 2007), usually then known as English ivy.The species is also notable for its rich folklore, and is still widely used in wreaths and celebrated in Christmas songs.
The taxonomy of ivy is complex, with disagreement around the recognition of subspecific taxa and confusion around the European native status of some horticultural taxa.Here, we follow Stace (2010), with our specimen belonging to the widespread diploid Hedera helix (syn.Hedera helix subsp.helix) which has 2n = 48, rather than the more westerly distributed tetraploid Hedera hibernica Poit.Here, we present the first high-quality ivy genome, which we anticipate being a valuable genomic resource for a range of future studies.These may include investigations into the biosynthetic pathway underlying the production of triterpenoid saponins, which has previously been studied in ivies using transcriptomic data (Sun et al., 2017).The species is also of interest for its developmental genetics (Schäffner & Nagl, 1979).The species undergoes a dramatic transition in leaf shape, with juveniles producing five-lobed leaves, while adults produce radially symmetrical ovate leaves (Metcalfe, 2005).Finally, ivy is the host plant of the parasitic ivy broomrape (Orobanche hederae Duby), and comparative genomic analyses could be used to investigate interactions in this obligate parasite-host system (Twyford, 2018).

Genome sequence report
The genome was sequenced from one Hedera helix specimen (Figure 1) collected from Petersham Common, Richmond, Surrey, UK (latitude 51.45, longitude -0.30).Using flow cytometry, the genome size (1C-value) was estimated to be 1.59 pg, equivalent to 1,550 Mb.A total of 29-fold coverage in Pacific Biosciences single-molecule HiFi long reads and 65-fold coverage in 10X Genomics read clouds were generated.Primary assembly contigs were scaffolded with chromosome conformation Hi-C data.Manual assembly curation corrected 276 missing joins or mis-joins and removed 15 haplotypic duplications, reducing the assembly length by 0.63% and the scaffold number by 79.25%, and increasing the scaffold N50 by 10.54%.
The final assembly has a total length of 1,199.4Mb in 55 sequence scaffolds with a scaffold N50 of 50.5 Mb (Table 1).Most (99.84%) of the assembly sequence was assigned to 24 chromosomal-level scaffolds.Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size (Figure 2-Figure 5; Table 2).There is a region of low confidence on Chromosome 1 at 15-26.1 Mb consisting of several repetitive scaffolds with uncertain orientation and order.The Hi-C data indicate there is a nested heterozygous inversion on Chromosome 3 covering the approximate region 19.7-35.7 Mb.On Chromosome 20, there is a heterozygous inversion between approximately 1.6-4.4Mb.The mitochondrial and chloroplast genomes were also assembled.
The assembly has a BUSCO v5.3.2 (Manni et al., 2021) completeness of 99.2% (single 57.2%, duplicated 42.0%) using  the eudicots_odb10 reference set.While not fully phased, the assembly deposited is of one haplotype.Contigs corresponding to the second haplotype have also been deposited.
The estimated Quality Value (QV) of the final assembly is 54.2 with k-mer completeness of 99.99%, and the assembly has a BUSCO v5.3.2 completeness of 99.2% (single = 57.2%,Metadata for specimens, spectral estimates, sequencing runs, contaminants and pre-curation assembly statistics can be found at https://links.tol.sanger.ac.uk/species/4052.

Sample acquisition, genome size estimation and nucleic acid extraction
A Hedera helix specimen (drHedHeli1) was collected from Petersham Common, Richmond, Surrey, UK (latitude 51.45, longitude -0.30) on 8 September 2020.The specimen was picked by hand from a beech woodland habitat by Maarten Christenhusz (Royal Botanic Gardens, Kew) collection number 9099.The specimen was identified by Maarten Christenhusz based on its morphology and preserved by freezing at -80°C.
A second specimen (drHedHeli8) was collected from the Royal Botanic Garden Edinburgh (Inverleith) (latitude 55.96, longitude -3.20) on 23 November 2020 by David Bell (Royal Botanic Garden Edinburgh).The specimen was identified by David Bell based on its morphology and flash-frozen in liquid nitrogen.This specimen was used for RNA sequencing.
The genome size was estimated by flow cytometry using the fluorochrome propidium iodide and following the 'one-step' method outlined in Pellicer et al. (2021).Specifically for this RNA was extracted from leaf tissue of drHedHeli8 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 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.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 (Harry, 2022).The mitochondrial and chloroplast genomes were assembled using MBG (Rautiainen & Marschall, 2021) from PacBio HiFi reads mapping to related genomes.A representative circular sequence was selected for each from the graph based on read coverage.
Table 3 contains a list of relevant software tool versions and sources.

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.
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

Shu-Miaw Chaw
Biodiversity Research Center Academia Sinica, Taipei City, Taipei City, Taiwan This manuscript reports the first reference genome sequence of Hedera helix L. (English ivy or common ivy).As English ivy and other introduced ivy species (have escaped from gardens) are now introduced (or probably invasive) aliens globally, and the taxonomy of ivy is complex.
Completion and annotation of a high-quality genome sequence of the common ivy will certainly be an important resource to understand not only the development of several morphological characters (such as leaf lobe and climbing habit), but also genes involved in invasiveness, biosynthesis of secondary metabolites as well as interaction mechanism with its parasitic plants.
To this end, this manuscript is well written, concise, and clear.It will be appealing to general readers.The methodology about assembly and its quality verification are correctly described with detailed explanation and logics.The figures and tables are excellently illustrated with high resolution and quality, though it is hard for me to understand Figure 3.I strongly recommend acceptance of this manuscript for indexing.

Yoshinori Fukasawa
Center for Center for Bioscience Research and Education, Utsunomiya University, Tochigi, Japan The authors present the genome assembly of common ivy, Hedera helix (hereafter H. helix), because it provides a comprehensive understanding of the genetic makeup of this species.The assembly is nearly 1.2 Gb in size and is scaffolded into 24 chromosomal pseudomolecules (55 sequence scaffolds in total) using multiple platforms.In addition, the mitochondrial and plastid genomes have also been assembled.This complete genome assembly could be used as a fundamental resource in studies of the common ivy.
From a technical perspective, the genome assembly process for H. helix was performed using a combination of long-read sequencing technologies, Hi-C scaffolding, and manual curation.The authors used Hifiasm for the initial assembly, which was then polished using 10X Genomics read data and FreeBayes variant calling.The assembly was then scaffolded using Hi-C data, and finally manual curation was performed using gEVAL, HiGlass, and Pretext.
One of the major challenges the researchers faced was the complexity of the genome such as the presence of haplotypic duplications, and the need for manual curation to correct errors in the assembly.Standard contiguity metrics such as N50 and BUSCO show convincing values.

Minor comments:
Polishing the HiFi assembly with short read data poses a risk of introducing technical errors, especially at this level of accuracy.To address this issue, a recent study utilized a hybrid variant calling model that can handle both PacBio HiFi and Illumina short reads simultaneously (Mc Cartney et al., 2022 1 ).It would be beneficial for the authors to provide a citation to support this approach or show the improvement from polishing in the assembly. 1.
Could you please specify which read set(s) were used for the k-mer computation in the QV calculation with Merqury?This detail seems to be missing.

2.
Could you clarify if the pseudomolecules extend from telomere to telomere, at least for some of them?This would be helpful to know.

Figure 1 .
Figure 1.Example images of the common ivy Hedera helix (not the sampled specimen) growing in secondary woodland in Edinburgh.a) juvenile foliage.b) mature foliage and fruits.Photos taken by Alex Twyford.

Figure 2 .
Figure 2. Genome assembly of Hedera helix, drHedHeli1.2: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 1,199,358,263 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 (62,841,030 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (50,454,882 and 40,242,199 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 eudicots_odb10 set is shown in the top right.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/drHedHeli1.2/dataset/CAMXCC02/snail.

Figure 3 .
Figure 3. Genome assembly of Hedera helix, drHedHeli1.2: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/drHedHeli1.2/dataset/CAMXCC02/blob.)

Figure 4 .
Figure 4. Genome assembly of Hedera helix, drHedHeli1.2: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/drHedHeli1.2/dataset/ CAMXCC02/cumulative.

Figure 5 .
Figure 5. Genome assembly of Hedera helix, drHedHeli1.2:Hi-C contact map.Hi-C contact map of the drHedHeli1.2assembly, 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=RqLbs2mdRkulj0bOy5MAQw.

Open Peer Review Current Peer Review Status: Version 1
This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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
Competing Interests: No competing interests were disclosed.Reviewer Expertise: Plant comparative genomicsI

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.
This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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 Competing Interests:
.Nat Methods.2022; 19 (6): 687-695 PubMed Abstract | Publisher Full Text No competing interests were disclosed.