The genome sequence of field madder, Sherardia arvensis L., 1753 (Rubiaceae)

We present a genome assembly from an individual Sherardia arvensis (field madder; Tracheophyta; Magnoliopsida; Gentianales; Rubiaceae). The genome sequence is 440.9 megabases in span. Most of the assembly is scaffolded into 11 chromosomal pseudomolecules. The mitochondrial and plastid genome assemblies have lengths of 203.98 kilobases and 152.73 kilobases in length, respectively.


Background
Easily overlooked, the field madder, Sherardia arvensis is a small, annual plant found in open, dry grassland, sheltered cliffs, sand dunes, arable fields, waste ground, waysides and verges.It is the only species in the genus Sherardia, which is distinguished from other Rubiaceae by the fused involucral bracts forming flower heads.The genus was named in honour of English botanist and consul William Sherard (1659-1728).Sherardia arvensis was formerly frequent, but is now much decreased in Britain and Ireland due to agricultural intensification since the 1950s (OABIF, 2022;Sutcliffe & Kay, 2000).Studies of the genetic variation within and between populations from different habitats found that a clear correlation between geographical and genetic distances could was only found for the indigenous populations studied and was absent in sites characterised by more intensive agricultural practices (Listl & Reisch, 2014).
Sherardia arvensis makes a mat of trailing, quadrangular stems up to 40 cm long.The leaves, approximately 1 cm in length, are placed in whorls of four to six and are roughly hairy.Flowers are grouped with up to three together in leaf axils surrounded by involucral bracts.The tiny (3 mm wide) petals are lilac-blue and fused into a funnel with four pointed lobes.
Flowers are pollinated by flies, but will also regularly self-pollinate.Fruits are composed of two indehiscent nutlets.
Field madder is found naturally throughout Europe and north Africa, and east into west and central Asia.It has been commonly introduced as an agricultural weed in other temperate regions of North and South America, East and South Africa, Bermuda, Taiwan, Hawaii, Australia, New Zealand and sub-Antarctic islands (POWO, 2022).In Britain and Ireland, it is likely that it is native only in western coastal habitats, whereas elsewhere it is an archaeophyte (OABIF, 2022).
The roots are reported to have been used by the ancient Celts as a source of a red or pink dye, but the quality is inferior to another species in the Rubiaceae -the dyer's madder (Rubia tinctorum;Moerman, 1998;Riley, 1997) which has been much more widely used (Biertümpfel & Wurl, 2009).
We here present the first complete genome of Sherardia arvensis.This will aid in understanding of how the native populations have spread across Britain and beyond.

Genome sequence report
The genome was sequenced from a specimen of Sherardia arvensis (Figure 1) collected from Teddington Lock, Surrey, UK (51.43, -0.33).Using flow cytometry, the genome size (1C-value) was estimated to be 0.60 pg, equivalent to 580 Mb.A total of 76-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 4 missing joins or mis-joins.The final assembly has a total length of 440.9 Mb in 11 sequence scaffolds with a scaffold N50 of 43.6 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 (99.91%) of the assembly sequence was assigned to 11 chromosomal-level scaffolds, which is consistent with the diploid chromosome count from British material reported for this species (i.e. 2n = 22;Maude, 1939).Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size (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 and plastid genomes were also assembled and can be found as contigs within the multifasta file of the genome submission.

Sample acquisition, genome size estimation and nucleic acid extraction
A specimen of Sherardia arvensis (specimen ID KDTOL10195, ToLID daSheArve1) was picked by hand from the edge  High Sensitivity Assay kit.Fragment size distribution was evaluated by running the sample on the FemtoPulse system.
RNA was extracted from leaf tissue of daSheArve1 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 core laboratory are publicly available on protocols.io(Denton et al., 2023).

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 leaf tissue of daSheArve1 using the Arima2 kit and sequenced on the Illumina NovaSeq 6000 instrument.

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 The authors used contemporary methods for the sequencing and assembly and provided detailed metrics of all aspects of their work along with open source, clear protocols.
The genome has not been annotated and will be submitted to the ENsembl team for annotation; it would be good to link the genome sequence and this publication to the annotation once it is generated.
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 genome biology 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 Sherardia arvensis, daSheArve1.1: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 441,296,019 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 (53,145,315 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (43,593,172 and 27,662,378 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/daSheArve1_1/dataset/daSheArve1_1/snail.

Figure 3 .
Figure 3. Genome assembly of Sherardia arvensis, daSheArve1.1: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/daSheArve1_1/dataset/daSheArve1_1/blob.

Figure 4 .
Figure 4. Genome assembly of Sherardia arvensis, daSheArve1.1: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/daSheArve1_1/dataset/daSheArve1_1/cumulative.

Figure 5 .
Figure 5. Genome assembly of Sherardia arvensis, daSheArve1.1:Hi-C contact map of the daSheArve1.1 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=Ny8eTdTwQGWpt06JQDWCLw.

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.

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