The genome sequence of the common toadflax, Linaria vulgaris Mill., 1768

We present a genome assembly from a Linaria vulgaris specimen (common toadflax; Streptophyta; Magnoliopsida; Lamiales; Plantaginaceae). The genome sequence is 760.5 megabases in span. Most of the assembly is scaffolded into six chromosomal pseudomolecules. Two mitochondrial genomes were assembled, which were 330.8 and 144.0 kilobases long. The plastid genome was also assembled and is 156.7 kilobases in length.


Background
Common toadflax, Linaria vulgaris Mill.(Plantaginaceae;2n = 2x = 12 (Henniges et al., 2022), is a short-lived herbaceous perennial plant.It is native to most of Europe to North and Central Asia, the Himalayas and eastern China (POWO, 2022), and was introduced to South Africa, northeast Asia and the temperate Americas, where it can be highly invasive (Sing & Peterson, 2011).It is widely distributed throughout Britain and Ireland, although it is less common in northern Scotland and western Ireland (Preston et al., 2002;Stace et al., 2019).Linaria vulgaris avoids acid soils but tolerates a wide range of habitats, such as open meadows and disturbed roadsides (Sing & Peterson, 2011;Stace et al., 2019).This species readily hybridises with other members of its genus (Stace et al., 2015;Ward et al., 2009).One of the best-studied Linaria hybrids in Britain and Ireland is L. vulgaris × repens, which is highly fertile and able to backcross with both parental species (Druce, 1896;Stace et al., 2015).
The foliage superficially resembles that of the flax (Linum L.), whence the names 'toadflax' and 'Linaria' derive (Gledhill, 2008).The zygomorphic flowers, borne on terminal racemes, are pale-yellow with orange centres (Figure 1A, B).Each flower contains a tubular nectar spur, which holds a reservoir of nectar that is accessible to long-tongued bumblebee pollinators (Newman & Thomson, 2005) (Figure 1C).Linaria vulgaris reproduces sexually, via bee-mediated pollination, and asexually, via vegetative propagation from adventitious buds which develop on the roots (Bakshi & Coupland, 1960).Seeds are dust-like and dispersed by wind.
Linaria vulgaris has been of particular interest to botanists for over 250 years, since Linnaeus described mutant toadflax flowers that displayed radial, instead of the typical bilateral, symmetry (Gustafsson, 1979;Linneaus, 1749).Later characterisation of these mutant flowers led to the first description of naturally occurring epigenetic mutations (epimutations) (Cubas et al., 1999).It is currently being developed as a model system for studying nectar spur development and floral evolution (Cullen et al., 2018), and has recently been genetically transformed via Agrobacterium-mediated transformation (Figure 1D).
We hope that the sequence provided here will contribute to the study of Linaria vulgaris as an emerging model for understanding the genetics of floral evolution and flower development.This whole genome sequence will also facilitate population genetic studies of natural toadflax populations.

Genome sequence report
The genome was sequenced from a specimen of Linaria vulgaris (Figure 1) collected from along the River Thames towpath in Kingston upon Thames, Surrey, UK (latitude 51.43, longitude -0.31).Using flow cytometry, the genome size (1C-value) was estimated to be 0.96 pg, equivalent to 940 Mb.A total of 28-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 69 missing joins or mis-joins and removed 58 haplotypic duplications, reducing the assembly length by 3.2% and the scaffold number by 59.8%, and decreasing the scaffold N50 by 17.75%.
The final assembly has a total length of 760.5 Mb in 41 sequence scaffolds with a scaffold N50 of 127.5 Mb (Table 1).Most (99.62%) of the assembly sequence was assigned to six chromosomal-level scaffolds.Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size (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.

Sample acquisition, genome size estimation and nucleic acid extraction
A Linaria vulgaris specimen (daLinVulg1) was collected from Kingston upon Thames, Surrey, UK (latitude 51.43, longitude -0.31) on 1 September 2020.The specimen was picked by hand by Maarten Christenhusz (Royal Botanic Gardens, Kew) from riparian vegetation on sand along the River Thames towpath.The specimen was identified based on its morphology by Maarten Christenhusz, and was preserved by freezing at -80°C.
The genome size was estimated by flow cytometry using the fluorochrome propidium iodide and following the 'one-step' method as outlined in Pellicer et al. (2021).Specifically for this species, the General Purpose Buffer (GPB) supplemented with 3% PVP and 0.08% (v/v) beta-mercaptoethanol was used for isolation of nuclei (Loureiro et al., 2007), and the internal calibration standard was Petroselinum crispum 'Champion Moss Curled' with an assumed 1C-value of 2,200 Mb (Obermayer et al., 2002).
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI).The daLinVulg1 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing.Leaf tissue was cryogenically disrupted to a fine powder using a Covaris cryoPREP Automated Dry RNA was extracted from flower tissue of daLinVulg1 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 the Agilent RNA 6000 Pico Kit and Eukaryotic Total RNA assay.

Sequencing
Pacific Biosciences HiFi circular consensus DNA sequencing libraries were constructed according to the manufacturers' instructions.Poly(A) RNA-Seq libraries were constructed  (Zhou et al., 2023).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) andPretext (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.
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

Xin Liu
State Key Laboratory of Agricultural Genomics, BGI (Beijing Genomics Institute)-Shenzhen, Shenzhen, China The data note by Maarten et al. described the established dataset of the common toadflax genome.The dataset is of good quality, with background information and methods well described.As a data note, I think it is acceptable and can be further used in future studies.
Meanwhile, I have two suggestions, in which I would recommend the authors to provide further information.First of all, the assembled genome size was substantially smaller than the estimated genome size (by flow cytometry).It would be better if the authors can provide further information (for example, estimation through kmer analysis), or maybe discussion on possible reason for the inconsistency here.Secondly, although I do not know whether it is required for the DToL project, I would suggest including gene annotation result in the dataset, which would make the dataset to be more complete and reusable.
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: genomics, plant genomics, bioinformatics 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 1 .
Figure 1.Flowers of Linaria vulgaris (not the sampled specimen).A. Flowers are borne on terminal racemes.B. Flowers are lobed and bilaterally symmetrical.C. Each flower possesses a single nectar spur.D Photograph of Linaria vulgaris expressing YFP following Agrobacterium-mediated transformation.

Figure 2 .
Figure 2. Genome assembly of Linaria vulgaris, daLinVulg1.1: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 760,456,579 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 (153,338,251 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (127,462,361 and 110,092,459 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/daLinVulg1.1/dataset/CAOJCB01/snail.

Figure 3 .
Figure 3. Genome assembly of Linaria vulgaris, daLinVulg1.1: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/daLinVulg1.1/dataset/CAOJCB01/blob.

Figure 4 .
Figure 4. Genome assembly of Linaria vulgaris, daLinVulg1.1: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/daLinVulg1.1/dataset/ CAOJCB01/cumulative.

Figure 5 .
Figure 5. Genome assembly of Linaria vulgaris, daLinVulg1.1:Hi-C contact map.Hi-C contact map of the daLinVulg1.1 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=Lroe3SmHRWOiVBKAhWt8bQ.

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:
The genome sequence is released openly for reuse.The Linaria vulgaris 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.The genome will be annotated using available RNA-Seq data and presented through the Ensembl pipeline at the European Bioinformatics Institute.Raw data and assembly accession identifiers are reported in Table1.No competing interests were disclosed.

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.