The genome sequence of common knotgrass, Polygonum aviculare L. (Polygonaceae)

We present a genome assembly from an individual Polygonum aviculare (common knotgrass; Eudicot; Magnoliopsida; Caryophyllales; Polygonaceae). The genome sequence is 351.6 megabases in span. Most of the assembly is scaffolded into 10 chromosomal pseudomolecules. The mitochondrial and plastid genome assemblies have lengths of 333.39 kilobases and 163.28 kilobases in length, respectively.


Background
The genome of the common knotgrass, Polygonum aviculare L., 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.
Polygonum aviculare is a procumbent to scrambling herbaceous annual with creeping stems up to 2 m long (Stace et al., 2019;Styles, 1962), but usually shorter.The stems are nodose, with an ochrea sheathing the stem in the axil of the lanceolate leaf.The five petals are white with a green midvein and often a pink tinge on the outside.The flowers are hermaphrodite and small, and considered to be largely self-pollinating (Styles, 1962).It has five to eight stamens and three fused carpels.The fruit is a dark brown, three-edged nut with a single seed that needs light to germinate.Seeds can lay dormant in the soil seedbank for decades (Courtney, 1968).The seeds were previously collected and consumed as porridge by Germanic peoples of western Europe (Nielsen et al., 2021).In herbal medicine, dried knotgrass is sometimes used in concoctions to treat gingivitis or other minor infections of the mouth or throat.It is also suggested as an alternative treatment for breast cancer (Habibi et al., 2011), but further study on the effective compounds is needed.Despite its name, common knotgrass is not a grass, but a member of the buckwheat family, Polygonaceae.Polygonum is derived from Greek poly (many) and gonu (knee-joint), referring to the swollen stem nodes.The epithet aviculare refers to small birds, said to consume the seeds.They are indeed the favourite food of partridges (Middleton & Chitty, 1937), among other birds.
It is widely distributed in Europe, North Africa and Asia, with populations as far south as Ethiopia and Myanmar (POWO, 2023), and it is common throughout Britain and Ireland with the exception of far north-western Scotland and the Scottish Highlands (Stroh et al., 2023).It has been extensively introduced outside its native range (North and South America, southern Africa, Tasmania, New Zealand), where it can be a colonising weed of fields, gardens and ruderal places (Meerts et al., 1998;POWO, 2023;Styles, 1962).
In Britain and Ireland it is found on open and disturbed ground, such as arable and other disturbed or trampled land, both rural and urban, soil, manure and waste heaps, paths and tracks, and has been recorded at elevations up to 550 m (with an outlier record of 670 m; Stroh et al., 2023).It can be a notorious weed in gardens, and can be problematic as an agricultural weed, particularly in open and spring-sown crops, such as spring beans, sugar beet, kale, linseed and potatoes (AHDB, 2008).
The taxonomy of Polygonum aviculare is complex, with Polygonum aviculare previously treated as an aggregate including taxa now recognised as distinct species such as P. boreale (Lange) Small (Stroh et al., 2023;Styles, 1962).Currently, three subspecies are accepted (POWO, 2023): P. aviculare subsp.buxiforme (Small) Costea & Tardif native to North America, P. aviculare subsp.neglectum (Besser) Arcang. in Siberia, the Caucasus, Greece, Italy and France, and the nominal, widespread P. aviculare subsp.aviculare, which is sampled here.However, some of these subspecies may also turn out to be independent species.More taxonomic study is needed.
From Britain and Ireland there are chromosome counts from four populations, with a reported chromosome number of 2n = 60 (Gornall & Bailey, 1993;Styles, 1962).Based on chromosome counts of 2n = 20 and 2n = 40 from other species in the genus, the inferred ploidy of Polygonum aviculare is hexaploid (Meerts et al., 1998;Styles, 1962).A previous isozyme study showed high levels of fixed heterozygosity in Polygonum aviculare consistent with the suggestion that it was an allopolyploid (Meerts et al., 1998).
In this paper we present a high-quality reference genome as a foundation resource for future studies on taxonomy, agronomy and potential medicinal applications of this herb.

Genome sequence report
The genome was sequenced from a specimen of Polygonum aviculare (Figure 1) collected from the Royal Botanic Gardens Kew (51.48, -0.29).Using flow cytometry, the genome size (1C-value) was estimated to be 0.93 pg, equivalent to 910 Mb.A total of 68-fold coverage in Pacific Biosciences single-molecule HiFi long reads and 51-fold coverage in 10X Genomics read clouds was generated.Primary assembly contigs were scaffolded with chromosome conformation Hi-C data.Manual assembly curation corrected 5 missing joins or mis-joins and removed 9 haplotypic duplications, reducing the assembly length by 8.16% and the scaffold number by 10.42%, and increasing the scaffold N50 by 5.85%.
The final assembly has a total length of 351.6 Mb in 86 sequence scaffolds with a scaffold N50 of 33.8 Mb (Table 1).The discrepancy between the genome assembly size, which is approximately a third of the flow cytometry estimate (above), suggests the individual sequenced here is a hexaploid with an estimated haploid genome size 303 Mb comprising 10 chromosomes.The snailplot 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 (98.06%) of the assembly sequence was assigned to 10 chromosomal-level scaffolds.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 a 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 Polygonum aviculare (specimen ID KDTOL10108, ToLID dcPolAvic1) was picked by hand as a weed from a bed from Royal Botanic Gardens Kew (latitude 51.48, longitude -0.29) on 8 September 2020.The was collected and identified by Maarten Christenhusz (Royal Botanic Gardens, Kew; collection no 9102) and then preserved by freezing at -80 °C.Herbarium voucher specimens collected from the same population as the sequenced specimen are lodged in the Herbarium of the Royal Botanic Gardens Kew (K) under accession number K001400691, and at the Royal Botanic Garden Edinburgh (E) (https://data.rbge.org.uk/herb/E01152775).
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).For this species, the General Purpose Buffer (GPB) supplemented with 3% PVP  was cryogenically disrupted using the Covaris cryoPREP ® Automated Dry Pulverizer (Narváez-Gómez et al., 2023).HMW DNA was extracted using the Automated Plant MagAttract v2 protocol (Todorovic et al., 2023a).HMW DNA was sheared into an average fragment size of 12-20 kb in a Megaruptor 3 system with speed setting 30 (Todorovic et al., 2023b).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 leaf tissue of dcPolAvic1 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.

Sequencing
Pacific Biosciences HiFi circular consensus and 10X Genomics read cloud 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), Illumina HiSeq 4000 (RNA-Seq) and Illumina NovaSeq 6000 (10X) instruments.Hi-C data were also  generated from leaf tissue of dcPolAvic1 the Arima2 kit and sequenced on the Illumina NovaSeq 6000 instrument.
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.The article describes clearly and concisely the generation of a new high-quality assembly for Polygonum aviculare.The text is easy to follow, the tables and figures are clear and informative and all the relevant methods and datasets are described appropriately.
The genome assembly presented here is a valuable resource in the context of the Darwin Tree of Life Project, which aims to sequence all species in Great Britain.
I have no concerns about the quality of this report and the datasets published here, and I would therefore recommend this article for indexing.

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 This paper describes the genome assembly of the herb common knotgrass.With an assembly size around 350Mb this represents a rather small plant genome.Sequencing and assembly follows the tree of life standards and is state-of-the-art.
Noteworthy, analyses indicate that knotgrass has a hexaploid genome...the assembly presented here represents a single haplotype.If the knotgrass genome is indeed an allopolyploid it is interesting to see that the subgenomes seem to be similar enough to mainly collapse into a single haplotype...although contigs from a second haplotype are presented and available as well.It would be interesting to get an idea about the degree of similarity between the different (in theory three) haplotypes of knotgrass.
The overall BUSCO score of around 93% complete is a little on the lower side...could this be attributed to the used BUSCO eudicots reference set and the place of Polygonum in the taxonomy?
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 genomics, bioinformatics I confirm that I have read this submission and believe that I have an appropriate level of

Figure 2 .
Figure 2. Genome assembly of Polygonum aviculare, dcPolAvic1.1: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 352,071,428 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 (42,788,684 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (33,827,450 and 28,844,451 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/CAKOHF01/dataset/CAKOHF01/snail.

Figure 3 .
Figure 3. Genome assembly of Polygonum aviculare, dcPolAvic1.1: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/CAKOHF01/dataset/CAKOHF01/blob.
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 assumed1C-value of 2,200 Mb (Obermayer et al., 2002).Protocols developed by the Wellcome Sanger Institute (WSI) Tree of Life core laboratory have been deposited on protocols.io(Denton et al., 2023).The workflow for high molecular weight (HMW) DNA extraction at the WSI includes a sequence of core procedures: sample preparation; sample homogenisation, DNA extraction, fragmentation, and clean-up.In sample preparation, the dcPolAvic1 sample was weighed and dissected on dry ice(Jay et al., 2023).For sample homogenisation, leaf

Figure 4 .
Figure 4. Genome assembly of Polygonum aviculare, dcPolAvic1.1: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/CAKOHF01/dataset/CAKOHF01/cumulative.

Figure 5 .
Figure 5. Genome assembly of Polygonum aviculare, dcPolAvic1.1:Hi-C contact map of the dcPolAvic1.1 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=FBNFeAZsTxm1pwpnccXwTg.

Competing Interests :
No competing interests were disclosed.Reviewer Expertise: Plant genomics I confirm that I have read this submission and believe that I have an appropriate level of expertise to that it is of an acceptable scientific standard.Reviewer Report 01 August 2024 https://doi.org/10.21956/wellcomeopenres.23236.r88617© 2024 Spannagl M. 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.Manuel Spannagl PGSB -Plant Genome and Systems Biology, Helmholtz Zentrum München, Helmholtz Zentrum München-German Research Center for Environmental Health, Neuherberg, Germany

Table 3
contains a list of relevant software tool versions and sources.Wellcome Sanger Institute -Legal and GovernanceThe 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 Prac- tice', 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.

Table 3 . Software tools: versions and sources. Software tool Version 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.