The genome sequence of the Roundleaf geranium, Geranium rotundifolium L. (Geraniales: Geraniaceae) - PMC Skip to main content An official website of the United States government Here's how you know Here's how you know Official websites use .gov A .gov website belongs to an official government organization in the United States. Secure .gov websites use HTTPS A lock ( Lock Locked padlock icon ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites. Search Log in Dashboard Publications Account settings Log out Search… Search NCBI Primary site navigation Search Logged in as: Dashboard Publications Account settings Log in Search PMC Full-Text Archive Search in PMC Journal List User Guide PERMALINK Copy As a library, NLM provides access to scientific literature. Inclusion in an NLM database does not imply endorsement of, or agreement with, the contents by NLM or the National Institutes of Health. Learn more: PMC Disclaimer | PMC Copyright Notice Wellcome Open Res . 2026 Mar 18;11:168. [Version 1] doi: 10.12688/wellcomeopenres.26116.1 Search in PMC Search in PubMed View in NLM Catalog Add to search The genome sequence of the Roundleaf geranium, Geranium rotundifolium L. (Geraniales: Geraniaceae) Maarten J M Christenhusz Maarten J M Christenhusz 1 Royal Botanic Gardens Kew, Richmond, England, UK 2 Curtin University, Perth, Western Australia, Australia Investigation, Resources Find articles by Maarten J M Christenhusz 1, 2 , Alex D Twyford Alex D Twyford 3 Royal Botanic Garden Edinburgh, Edinburgh, Scotland, UK 4 The University of Edinburgh, Edinburgh, Scotland, UK Writing – Original Draft Preparation, Writing – Review & Editing Find articles by Alex D Twyford 3, 4 ; Royal Botanic Gardens Kew Genome Acquisition Lab ; Plant Genome Sizing Collective ; Wellcome Sanger Institute Tree of Life Management, Samples and Laboratory team ; Wellcome Sanger Institute Scientific Operations: Sequencing Operations ; Wellcome Sanger Institute Tree of Life Core Informatics team ; Tree of Life Core Informatics collective ; Darwin Tree of Life Consortium a Author information Article notes Copyright and License information 1 Royal Botanic Gardens Kew, Richmond, England, UK 2 Curtin University, Perth, Western Australia, Australia 3 Royal Botanic Garden Edinburgh, Edinburgh, Scotland, UK 4 The University of Edinburgh, Edinburgh, Scotland, UK a Email: [email protected] No competing interests were disclosed. Roles Maarten J M Christenhusz : Investigation, Resources Alex D Twyford : Writing – Original Draft Preparation, Writing – Review & Editing Accepted 2026 Apr 21; Collection date 2026. Copyright: © 2026 Christenhusz MJM et al. This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. PMC Copyright notice PMCID: PMC13096780 PMID: 42021764 Abstract We present a genome assembly of Geranium rotundifolium (Roundleaf geranium; Streptophyta; Magnoliopsida; Geraniales; Geraniaceae). The genome sequence has a total length of 497.00 megabases. Most of the assembly (97.57%) is scaffolded into 13 chromosomal pseudomolecules. The mitochondrial sequence has a length of 335.81 kilobases and the plastid genome assembly has a length of 169.49 kilobases. Gene annotation of this assembly on Ensembl identified 29 331 protein-coding genes. This assembly was generated as part of the Darwin Tree of Life project, which produces reference genomes for eukaryotic species found in Britain and Ireland. Keywords: Geranium rotundifolium; Roundleaf geranium; genome sequence; chromosomal; Geraniales Species taxonomy Eukaryota; Viridiplantae; Streptophyta; Streptophytina; Embryophyta; Tracheophyta; Euphyllophyta; Spermatophyta; Magnoliopsida; Mesangiospermae; eudicotyledons; Gunneridae; Pentapetalae; rosids; malvids; Geraniales; Geraniaceae; Geranium ; Geranium rotundifolium L. (NCBI:txid379955). Background The round-leaved crane’s-bill, G. rotundifolium L. ( Figure 1 ), is an annual herbaceous plant species that possesses abundant glandular hairs, has small pink flowers, and leaves that are kidney shaped in outline with relatively shallow lobes ( Stace et al. , 2019 ). It is locally abundant in the south of England and south Wales, while it is virtually absent from northern Scotland and much of Ireland ( Stroh et al. , 2023 ). However, the species has experienced a marked northwards expansion in the past ~50 years, perhaps associated with climate change. Outside of Britain and Ireland, its native range includes much of Europe, North Africa and Western Asia, and it has naturalised in many locations including in North America, South America and South Africa ( Royal Botanic Gardens, Kew, 2025 ). G. rotundifolium is typically found growing in walls and dry roadside banks, as well as a weed in disturbed sites and waste ground. Figure 1. Photograph of Geranium rotundifolium by Alvesgaspar . Open in a new tab The species belongs to the Geraniaceae, and is a diploid with 2 n = 26 ( Henniges et al. , 2022 ). Although numerous genomes exist for the family Geraniaceae, this assembly provides the first chromosomally complete sequence for G. rotundifolium , enabling comparative analyses. This genome was assembled using the Tree of Life pipeline from a specimen collected in Teddington Lock, Richmond, Surrey, UK. Methods Sample acquisition, flow cytometry and DNA barcoding A specimen of G. rotundifolium (specimen ID KDTOL10167, ToLID drGerRotu1) was used for genome sequencing. It was collected from Teddington Lock, Richmond, Surrey, UK (latitude 51.4303, longitude −0.319) on 2021-04-19. The specimen was collected and identified by Maarten J. M. Christenhusz (Royal Botanic Gardens Kew). The same specimen was used for RNA sequencing. Metadata collection followed the recommended standards of the Darwin Tree of Life project ( Lawniczak et al. , 2022 ). The genome size was estimated by flow cytometry following the ‘one-step’ method outlined in Pellicer et al . (2021) and using propidium iodide as the fluorochrome. 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 (Mill) Nyman ex A.E.Hill ‘Champion Moss Curled’ with an assumed 1C-value of 2 200 Mb ( Obermayer et al. , 2002 ). The initial identification was verified by an additional DNA barcoding process according to the framework developed by Twyford et al . (2024) . Part of the plant specimen was preserved in silica gel desiccant ( Chase & Hills, 1991 ). DNA extracted from the dried plant was amplified by PCR for standard barcode markers, with the amplicons sequenced and compared to public sequence databases including GenBank and the Barcode of Life Database (BOLD) ( Ratnasingham & Hebert, 2007 ). Following whole genome sequence generation, the relevant DNA barcode region was also used alongside the initial barcoding data for sample tracking at the WSI ( Twyford et al. , 2024 ). The standard operating procedures for Darwin Tree of Life barcoding are available on protocols.io . Nucleic acid extraction Protocols for high molecular weight (HMW) DNA extraction developed at the Wellcome Sanger Institute (WSI) Tree of Life Core Laboratory are available on protocols.io ( Howard et al. , 2025 ). The drGerRotu1 sample was weighed and triaged to determine the appropriate extraction protocol. Tissue from the leaf was homogenised by cryogenic disruption using the Covaris cryoPREP ® Automated Dry Pulverizer. HMW DNA was extracted using the Plant Organic Extraction protocol. We used centrifuge-mediated fragmentation to produce DNA fragments in the 8–10 kb range, following the Covaris g-TUBE protocol for ultra-low input (ULI). Sheared DNA was purified by automated SPRI (solid-phase reversible immobilisation), using AMPure PB beads (Pacific Biosciences) and the Thermo Fisher KingFisher™ Apex 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 using the Qubit dsDNA High Sensitivity Assay kit. Fragment size distribution was evaluated by running the sample on the FemtoPulse system. For this sample, the final post-shearing DNA had a Qubit concentration of 1.95 ng/μL and a yield of 760.50 ng. RNA was extracted from leaf tissue of drGerRotu1 in the Tree of Life Laboratory at the WSI using the RNA Extraction: Automated MagMax™ mir Vana protocol . 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. PacBio HiFi library preparation and sequencing Library preparation and sequencing were performed at the WSI Scientific Operations core. Prior to library preparation, the DNA was fragmented to ~10 kb. Ultra-low-input (ULI) libraries were prepared using the PacBio SMRTbell ® Express Template Prep Kit 2.0 and gDNA Sample Amplification Kit. Samples were normalised to 20 ng DNA. Single-strand overhang removal, DNA damage repair, and end-repair/A-tailing were performed according to the manufacturer’s instructions, followed by adapter ligation. A 0.85× pre-PCR clean-up was carried out with Promega ProNex beads. The DNA was evenly divided into two aliquots for dual PCR (reactions A and B), both following the manufacturer’s protocol. A 0.85× post-PCR clean-up was performed with ProNex beads. DNA concentration was measured using a Qubit Fluorometer v4.0 (Thermo Fisher Scientific) with the Qubit HS Assay Kit, and fragment size was assessed on an Agilent Femto Pulse Automated Pulsed Field CE Instrument (Agilent Technologies) using the gDNA 55 kb BAC analysis kit. PCR reactions A and B were then pooled, ensuring a total mass of ≥500 ng in 47.4 μl. The pooled sample underwent another round of DNA damage repair, end-repair/A-tailing, and hairpin adapter ligation. A 1× clean-up was performed with ProNex beads, followed by DNA quantification using the Qubit and fragment size analysis using the Agilent Femto Pulse. Size selection was performed on the Sage Sciences PippinHT system, with target fragment size determined by Femto Pulse analysis (typically 4–9 kb). Size-selected libraries were cleaned with 1.0× ProNex beads and normalised to 2 nM before sequencing. The sample was sequenced using the Sequel IIe system (Pacific Biosciences, California, USA). The concentration of the library loaded onto the Sequel IIe was in the range 40–135 pM. The SMRT link software, a PacBio web-based end-to-end workflow manager, was used to set-up and monitor the run, and to perform primary and secondary analysis of the data upon completion. Hi-C Sample preparation and crosslinking Hi-C data were generated from the leaf tissue of drGerRotu1 using the Arima-HiC v2 kit (Arima Genomics). Tissue was finely ground using the Covaris cryoPREP Dry Pulverizer (Covaris), and then subjected to nuclei isolation. Nuclei were isolated using a modified protocol based on the Qiagen QProteome Cell Compartment Kit (Qiagen), in which only the Lysis and CE2 buffers were used, with QIAshredder spin columns. After isolation, nuclei were fixed using formaldehyde to a final concentration of 2% to crosslink the DNA. The crosslinked DNA was then digested and biotinylated according to the manufacturer’s instructions. A clean-up step was performed with SPRIselect beads before library preparation. DNA concentration was quantified using the Qubit Fluorometer v4.0 (Thermo Fisher Scientific) and the Qubit HS Assay Kit, following the manufacturer’s instructions. Hi-C library preparation and sequencing Biotinylated DNA constructs were fragmented using a Covaris E220 sonicator and size selected to 400–600 bp using SPRISelect beads. DNA was enriched with Arima-HiC v2 kit Enrichment beads. End repair, A-tailing, and adapter ligation were carried out with the NEBNext Ultra II DNA Library Prep Kit (New England Biolabs), following a modified protocol where library preparation occurs while DNA remains bound to the Enrichment beads. Library amplification was performed using KAPA HiFi HotStart mix and a custom Unique Dual Index (UDI) barcode set (Integrated DNA Technologies). Depending on sample concentration and biotinylation percentage determined at the crosslinking stage, libraries were amplified with 10–16 PCR cycles. Post-PCR clean-up was performed with SPRISelect beads. Libraries were quantified using the AccuClear Ultra High Sensitivity dsDNA Standards Assay Kit (Biotium) and a FLUOstar Omega plate reader (BMG Labtech). Prior to sequencing, libraries were normalised to 10 ng/μL. Normalised libraries were quantified again to create equimolar and/or weighted 2.8 nM pools. Pool concentrations were checked using the Agilent 4200 TapeStation (Agilent) with High Sensitivity D500 reagents before sequencing. Sequencing was performed using paired-end 150 bp reads on the Illumina NovaSeq 6000. RNA library preparation and sequencing Libraries were prepared using the NEBNext ® Ultra™ II Directional RNA Library Prep Kit for Illumina (New England Biolabs), following the manufacturer’s instructions. Poly(A) mRNA in the total RNA solution was isolated using oligo (dT) beads, converted to cDNA, and uniquely indexed; 14 PCR cycles were performed. Libraries were size-selected to produce fragments between 100–300 bp. Libraries were quantified, normalised, pooled to a final concentration of 2.8 nM, and diluted to 150 pM for loading. Sequencing was carried out on the Illumina NovaSeq X to generate 150-bp paired-end reads. Genome assembly Prior to assembly of the PacBio HiFi reads, a database of k -mer counts ( k = 31) was generated from the filtered reads using FastK . GenomeScope2 ( Ranallo-Benavidez et al. , 2020 ) was used to analyse the k -mer frequency distributions, providing estimates of genome size, heterozygosity, and repeat content. The HiFi reads were assembled using Hifiasm ( Cheng et al. , 2021 ) with the --primary option. The Hi-C reads ( Rao et al. , 2014 ) were mapped to the primary contigs using bwa-mem2 ( Vasimuddin et al. , 2019 ), and the contigs were scaffolded in YaHS ( Zhou et al. , 2023 ) with the --break option for handling potential misassemblies. The scaffolded assemblies were evaluated using Gfastats ( Formenti et al. , 2022 ), BUSCO ( Manni et al. , 2021 ) and MERQURY.FK ( Rhie et al. , 2020 ). The organelle genomes were assembled using OATK [zhou2025Oatk]. Assembly curation The assembly was decontaminated using the Assembly Screen for Cobionts and Contaminants ( ASCC ) pipeline. TreeVal was used to generate the flat files and maps for use in curation. Manual curation was conducted primarily in PretextView and HiGlass ( Kerpedjiev et al. , 2018 ). Scaffolds were visually inspected and corrected as described by Howe et al . (2021) . Manual corrections included eight breaks, 14 joins, and removal of one haplotypic duplication. This reduced the scaffold count by 1.0%. The curation process is documented at https://gitlab.com/wtsi-grit/rapid-curation . PretextSnapshot was used to generate a Hi-C contact map of the final assembly. Assembly quality assessment The Merqury.FK tool ( Rhie et al. , 2020 ) was run in a Singularity container ( Kurtzer et al. , 2017 ) to evaluate k -mer completeness and assembly quality for the primary and alternate haplotypes using the k -mer databases ( k = 31) computed prior to genome assembly. The analysis outputs included assembly QV scores and completeness statistics. The genome was analysed using the BlobToolKit pipeline , a Nextflow implementation of the earlier Snakemake version ( Challis et al. , 2020 ). The pipeline aligns PacBio reads using minimap2 ( Li, 2018 ) and SAMtools ( Danecek et al. , 2021 ) to generate coverage tracks. It runs BUSCO ( Manni et al. , 2021 ) using lineages identified from NCBI Taxonomy ( Schoch et al. , 2020 ). For the three domain-level lineages, BUSCO genes are aligned to the UniProt Reference Proteomes database ( Bateman et al. , 2023 ) using DIAMOND blastp ( Buchfink et al. , 2021 ). The genome is divided into chunks based on the density of BUSCO genes from the closest taxonomic lineage, and each chunk is aligned to the UniProt Reference Proteomes database with DIAMOND blastx. Sequences without hits are chunked using seqtk and aligned to the NT database with blastn ( Altschul et al. , 1990 ). The BlobToolKit suite consolidates all outputs into a blobdir for visualisation. The BlobToolKit pipeline was developed using nf-core tooling ( Ewels et al. , 2020 ) and MultiQC ( Ewels et al. , 2016 ), with package management via Conda and Bioconda ( Grüning et al. , 2018 ), and containerisation through Docker ( Merkel, 2014 ) and Singularity ( Kurtzer et al. , 2017 ). Genome sequence report Sequence data The genome of a specimen of G. rotundifolium was sequenced using Pacific Biosciences single-molecule HiFi long reads, generating 44.27 Gb (gigabases) from 4.79 million reads, which were used to assemble the genome. GenomeScope2.0 analysis estimated the haploid genome size at 503.13 Mb, with a heterozygosity of 0.33% and repeat content of 37.85% ( Figure 2 ). Using flow cytometry, the genome size (1C-value) of the sample was estimated to be 0.65 pg, equivalent to 630.00 Mb. These estimates guided expectations for the assembly. Based on the estimated genome size, the sequencing data provided approximately 80× coverage. Hi-C sequencing produced 106.75 Gb from 706.93 million reads, which were used to scaffold the assembly. RNA sequencing data were also generated and are available in public sequence repositories. Table 1 summarises the specimen and sequencing details. Figure 2. Frequency distribution of k -mers generated using GenomeScope2. Open in a new tab The plot shows observed and modelled k -mer spectra, providing estimates of genome size, heterozygosity, and repeat content based on unassembled sequencing reads. Table 1. Specimen and sequencing data for BioProject PRJEB69504. Platform PacBio HiFi Hi-C RNA-seq ToLID drGerRotu1 drGerRotu1 drGerRotu1 Specimen ID KDTOL10167 KDTOL10167 KDTOL10167 BioSample (source individual) SAMEA9143062 SAMEA9143062 SAMEA9143062 BioSample (tissue) SAMEA9143830 SAMEA9143830 SAMEA9143830 Tissue leaf leaf leaf Instrument Sequel IIe Illumina NovaSeq 6000 Illumina NovaSeq X Run accessions ERR12303938; ERR12303939 ERR12318584 ERR13493917 Read count total 4.79 million 706.93 million 97.54 million Base count total 44.27 Gb 106.75 Gb 14.73 Gb Open in a new tab Assembly statistics The primary haplotype was assembled, and contigs corresponding to an alternate haplotype were also deposited in INSDC databases. The final assembly has a total length of 497.00 Mb in 496 scaffolds, with 698 gaps, and a scaffold N50 of 39.7 Mb ( Table 2 ). Table 2. Genome assembly statistics. Assembly name drGerRotu1.1 Assembly accession GCA_963920655.1 Alternate haplotype accession GCA_963920715.1 Assembly level chromosome Span (Mb) 497.00 Number of chromosomes 13 Number of contigs 1 194 Contig N50 1.1 Mb Number of scaffolds 496 Scaffold N50 39.7 Mb Organelles Mitochondrion: 335.81 kb; Plastid: 169.49 kb Open in a new tab Most of the assembly sequence (97.57%) was assigned to 13 chromosomal-level scaffolds. These chromosome-level scaffolds, confirmed by Hi-C data, are named according to size ( Figure 3 ; Table 3 ). Figure 3. Hi-C contact map of the Geranium rotundifolium genome assembly. Open in a new tab Assembled chromosomes are shown in order of size and labelled along the axes, with a megabase scale shown below. The plot was generated using PretextSnapshot. Table 3. Chromosomal pseudomolecules in the primary genome assembly of Geranium rotundifolium drGerRotu1. INSDC accession Molecule Length (Mb) GC% OY987311.1 1 48.13 39 OY987312.1 2 46.77 39.50 OY987313.1 3 41.76 38 OY987314.1 4 40.76 38.50 OY987315.1 5 40.25 39.50 OY987316.1 6 39.70 38 OY987317.1 7 38.16 38.50 OY987318.1 8 37.16 39 OY987319.1 9 36.52 39 OY987320.1 10 31.32 38 OY987321.1 11 28.59 38 OY987322.1 12 28.13 38.50 OY987323.1 13 27.66 38.50 Open in a new tab The mitochondrial genome (length 335.81 kb, OY987324.1 ) and plastid genome (length 169.49 kb, OY987325.1 ) were also assembled. These sequences are included as contigs in the multifasta file of the genome submission and as standalone records. Assembly quality metrics The combined primary and alternate assemblies achieve an estimated QV of 53.4. The k -mer completeness is 99.20% for the primary assembly, 5.12% for the alternate haplotype, and 99.23% for the combined assemblies ( Figure 4 ). Figure 4. Evaluation of k -mer completeness using MerquryFK. Open in a new tab This plot illustrates the recovery of k -mers from the original read data in the final assemblies. The horizontal axis represents k -mer multiplicity, and the vertical axis shows the number of k -mers. The black curve represents k -mers that appear in the reads but are not assembled. The green curve corresponds to k -mers shared by both haplotypes, and the red and blue curves show k -mers found only in one of the haplotypes. BUSCO v.5.5.0 analysis using the eudicots_odb10 reference set ( n = 2 326) identified 94.8% of the expected gene set (single = 80.0%, duplicated = 14.8%). The snail plot in Figure 5 summarises the scaffold length distribution and other assembly statistics for the primary assembly. The blob plot in Figure 6 shows the distribution of scaffolds by GC proportion and coverage. Figure 5. Assembly metrics for drGerRotu1.1. Open in a new tab The BlobToolKit snail plot provides an overview of assembly metrics and BUSCO gene completeness. The circumference represents the length of the whole genome sequence, and the main plot is divided into 1 000 bins around the circumference. The outermost blue tracks display the distribution of GC, AT, and N percentages across the bins. Scaffolds are arranged clockwise from longest to shortest and are depicted in dark grey. The longest scaffold is indicated by the red arc, and the deeper orange and pale orange arcs represent the N50 and N90 lengths. A light grey spiral at the centre shows the cumulative scaffold count on a logarithmic scale. A summary of complete, fragmented, duplicated, and missing BUSCO genes in the eudicots_odb10 set is presented at the top right. An interactive version of this figure can be accessed on the BlobToolKit viewer . Figure 6. BlobToolKit blob plot for drGerRotu1.1. Open in a new tab The plot shows base coverage (vertical axis) and GC content (horizontal axis). The circles represent scaffolds, with the size proportional to scaffold length and the colour representing phylum membership. The histograms along the axes display the total length of sequences distributed across different levels of coverage and GC content. An interactive version of this figure is available on the BlobToolKit viewer . Table 4 lists the assembly metric benchmarks adapted from Rhie et al. (2021) and the Earth BioGenome Project Report on Assembly Standards September 2024 . The EBP metric calculated for the primary assembly is 6.C.Q54 , meeting the recommended reference standard. Table 4. Earth Biogenome Project summary metrics for the Geranium rotundifolium assembly. Measure Value Benchmark EBP summary (primary) 6.C.Q54 6.C.Q40 Contig N50 length 1.10 Mb ≥ 1 Mb Scaffold N50 length 39.70 Mb = chromosome N50 Consensus quality (QV) Primary: 54.0; alternate: 48.5; combined: 53.4 ≥ 40 k -mer completeness Primary: 99.20%; alternate: 5.12%; combined: 99.23% ≥ 95% BUSCO C:100.0% [S:60.0%, D:40.0%], F:0.0%, M:0.0%, n:255 S > 90%; D < 5% Percentage of assembly assigned to chromosomes 97.57% ≥ 90% Open in a new tab Notes: The EBP summary uses log10(Contig N50); chromosome-level (C) or log10(Scaffold N50); Q (Merqury QV). BUSCO: C = complete; S = single-copy; D = duplicated; F = fragmented; M = missing; n = orthologues. Genome annotation report The G. rotundifolium genome assembly (GCA_963920655.1) was annotated by Ensembl at the European Bioinformatics Institute (EBI). This annotation includes 48 755 transcribed mRNAs from 29 331 protein-coding and 9 228 non-coding genes. The average transcript length is 2 941.21 bp, with an average of 1.26 coding transcripts per gene and 5.03 exons per transcript. For further information about the annotation, please refer to the annotation page on Ensembl. Author information • Members of the Royal Botanic Gardens Kew Genome Acquisition Lab • Members of the Plant Genome Sizing collective • Members of the Darwin Tree of Life Barcoding collective • Members of the Wellcome Sanger Institute Tree of Life Management, Samples and Laboratory team • Members of Wellcome Sanger Institute Scientific Operations – Sequencing Operations • Members of the Wellcome Sanger Institute Tree of Life Core Informatics team • Members of the Tree of Life Core Informatics collective • Members of the Darwin Tree of Life Consortium 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 . 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 • Legality of collection, transfer and use (national and international) Each 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. Funding Statement This work was supported by Wellcome through core funding to the Wellcome Sanger Institute (220540) and the Darwin Tree of Life Discretionary Award [218328, <a href=https://doi.org/10.35802/218328>https://doi.org/10.35802/218328 </a>]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. [version 1; peer review: 2 approved] Data availability European Nucleotide Archive: G. rotundifolium (round-leaved crane’s-bill). Accession number PRJEB69504 . The genome sequence is released openly for reuse. The G. rotundifolium genome sequencing initiative is part of the Darwin Tree of Life Project (PRJEB40665) and Sanger Institute Tree of Life Programme (PRJEB43745). 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 Tables 1 and 2 . Pipelines used for genome assembly at the WSI Tree of Life are available at https://pipelines.tol.sanger.ac.uk/pipelines . Table 5 lists software versions used in this study. Table 5. Software versions and sources. Software Version Source BEDTools 2.30.0 https://github.com/arq5x/bedtools2 BLAST 2.14.0 ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/ BlobToolKit 4.3.9 https://github.com/blobtoolkit/blobtoolkit BUSCO 5.5.0 https://gitlab.com/ezlab/busco bwa-mem2 2.2.1 https://github.com/bwa-mem2/bwa-mem2 Cooler 0.8.11 https://github.com/open2c/cooler DIAMOND 2.1.8 https://github.com/bbuchfink/diamond fasta_windows 0.2.4 https://github.com/tolkit/fasta_windows FastK 1.1 https://github.com/thegenemyers/FASTK GenomeScope2.0 2.0.1 https://github.com/tbenavi1/genomescope2.0 Gfastats 1.3.6 https://github.com/vgl-hub/gfastats GoaT CLI 0.2.5 https://github.com/genomehubs/goat-cli Hifiasm 0.19.5-r587 https://github.com/chhylp123/hifiasm HiGlass 1.13.4 https://github.com/higlass/higlass MerquryFK 1.1.2 https://github.com/thegenemyers/MERQURY.FK Minimap2 2.24-r1122 https://github.com/lh3/minimap2 Oatk 0.9 https://github.com/c-zhou/oatk MultiQC 1.14; 1.17 and 1.18 https://github.com/MultiQC/MultiQC Nextflow 23.04.1 https://github.com/nextflow-io/nextflow PretextSnapshot 0.0.5 https://github.com/sanger-tol/PretextSnapshot PretextView 0.2.5 https://github.com/sanger-tol/PretextView samtools 1.19.2 https://github.com/samtools/samtools sanger-tol/ascc 0.1.0 https://github.com/sanger-tol/ascc sanger-tol/blobtoolkit 0.4.0 https://github.com/sanger-tol/blobtoolkit Seqtk 1.3 https://github.com/lh3/seqtk Singularity 3.9.0 https://github.com/sylabs/singularity TreeVal 1.2.0 https://github.com/sanger-tol/treeval YaHS 1.2a.2 https://github.com/c-zhou/yahs Open in a new tab References Altschul SF, Gish W, Miller W, et al. : Basic local alignment search tool. J. Mol. Biol. 1990;215(3):403–410. 10.1016/S0022-2836(05)80360-2 [ DOI ] [ PubMed ] [ Google Scholar ] Bateman A, Martin M-J, Orchard S, et al. : UniProt: The Universal Protein Knowledgebase in 2023. Nucleic Acids Res. 2023;51(D1):D523–D531. 10.1093/nar/gkac1052 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Buchfink B, Reuter K, Drost H-G: Sensitive protein alignments at tree-of-life scale using DIAMOND. Nat. Methods. 2021;18(4):366–368. 10.1038/s41592-021-01101-x [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Challis R, Richards E, Rajan J, et al. : BlobToolKit – interactive quality assessment of genome assemblies. G3 Genes|Genomes|Genetics. 2020;10(4):1361–1374. 10.1534/g3.119.400908 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Chase MW, Hills HH: Silica gel: An ideal material for field preservation of leaf samples for DNA studies. Taxon. 1991;40(2):215–220. 10.2307/1222975 [ DOI ] [ Google Scholar ] Cheng H, Concepcion GT, Feng X, et al. : Haplotype-resolved de novo assembly using phased assembly graphs with Hifiasm. Nat. Methods. 2021;18(2):170–175. 10.1038/s41592-020-01056-5 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Danecek P, Bonfield JK, Liddle J, et al. : Twelve years of SAMtools and BCFtools. GigaScience. 2021;10(2). 10.1093/gigascience/giab008 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Ewels P, Magnusson M, Lundin S, et al. : MultiQC: Summarize analysis results for multiple tools and samples in a single report. Bioinformatics. 2016;32(19):3047–3048. 10.1093/bioinformatics/btw354 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Ewels PA, Peltzer A, Fillinger S, et al. : The nf-core framework for community-curated bioinformatics pipelines. Nat. Biotechnol. 2020;38(3):276–278. 10.1038/s41587-020-0439-x [ DOI ] [ PubMed ] [ Google Scholar ] Formenti G, Abueg L, Brajuka A, et al. : Gfastats: Conversion, evaluation and manipulation of genome sequences using assembly graphs. Bioinformatics. 2022;38(17):4214–4216. 10.1093/bioinformatics/btac460 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Grüning B, Dale R, Sjödin A, et al. : Bioconda: Sustainable and comprehensive software distribution for the life sciences. Nat. Methods. 2018;15(7):475–476. 10.1038/s41592-018-0046-7 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Henniges MC, Powell RF, Mian S, et al. : A taxonomic, genetic and ecological data resource for the vascular plants of Britain and Ireland. Sci. Data. 2022;9:1–8. 10.1038/s41597-022-01403-7 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Howard C, Denton A, Jackson B, et al. : On the path to reference genomes for all biodiversity: Lessons learned and laboratory protocols created in the Sanger Tree of Life core laboratory over the first 2000 species. bioRxiv. 2025. 10.1101/2025.04.11.648334 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Howe K, Chow W, Collins J, et al. : Significantly improving the quality of genome assemblies through curation. GigaScience. 2021;10(1). 10.1093/gigascience/giaa153 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Kerpedjiev P, Abdennur N, Lekschas F, et al. : HiGlass: Web-based visual exploration and analysis of genome interaction maps. Genome Biol. 2018;19(1):125. 10.1186/s13059-018-1486-1 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Kurtzer GM, Sochat V, Bauer MW: Singularity: Scientific containers for mobility of compute. PLoS One. 2017;12(5):e0177459. 10.1371/journal.pone.0177459 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Lawniczak MKN, Davey RP, Rajan J, et al. : Specimen and sample metadata standards for biodiversity genomics: A proposal from the Darwin Tree of Life project. Wellcome Open Res. 2022;7:187. 10.12688/wellcomeopenres.17605.1 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Li H: Minimap2: Pairwise alignment for nucleotide sequences. Bioinformatics. 2018;34(18):3094–3100. 10.1093/bioinformatics/bty191 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Loureiro J, Rodriguez E, Doležel J, et al. : Two new nuclear isolation buffers for plant DNA flow cytometry: A test with 37 species. Ann. Bot. 2007;100(4):875–888. 10.1093/aob/mcm152 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Manni M, Berkeley MR, Seppey M, et al. : BUSCO update: Novel and streamlined workflows along with broader and deeper phylogenetic coverage for scoring of eukaryotic, prokaryotic, and viral genomes. Mol. Biol. Evol. 2021;38(10):4647–4654. 10.1093/molbev/msab199 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Merkel D: Docker: Lightweight Linux containers for consistent development and deployment. Linux J. 2014;2014(239). 10.5555/2600239.2600241 [ DOI ] [ Google Scholar ] Obermayer R, Leitch IJ, Hanson L, et al. : Nuclear DNA C-values in 30 species double the familial representation in pteridophytes. Ann. Bot. 2002;90(2):209–217. 10.1093/aob/mcf167 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Pellicer J, Powell RF, Leitch IJ: The application of flow cytometry for estimating genome size, ploidy level endopolyploidy, and reproductive modes in plants. Besse P, editor. Methods in Molecular Biology. New York, NY:2021; Vol.2222:325–61. [ DOI ] [ PubMed ] [ Google Scholar ] Ranallo-Benavidez TR, Jaron KS, Schatz MC: GenomeScope 2.0 and Smudgeplot for reference-free profiling of polyploid genomes. Nat. Commun. 2020;11(1):1432. 10.1038/s41467-020-14998-3 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Rao SSP, Huntley MH, Durand NC, et al. : A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping. Cell. 2014;159(7):1665–1680. 10.1016/j.cell.2014.11.021 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Ratnasingham S, Hebert PDN: BOLD: The Barcode of Life Data System. Mol. Ecol. Notes. 2007;7(3):355–364. 10.1111/j.1471-8286.2007.01678.x http://www.barcodinglife.org [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Rhie A, McCarthy SA, Fedrigo O, et al. : Towards complete and error-free genome assemblies of all vertebrate species. Nature. 2021;592(7856):737–746. 10.1038/s41586-021-03451-0 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Rhie A, Walenz BP, Koren S, et al. : Merqury: Reference-free quality, completeness, and phasing assessment for genome assemblies. Genome Biol. 2020;21(1). 10.1186/s13059-020-02134-9 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Royal Botanic Gardens, Kew: POWO: Plants of the World Online. 2025. Reference Source Schoch CL, Ciufo S, Domrachev M, et al. : NCBI taxonomy: A comprehensive update on curation, resources and tools. Database. 2020;2020:baa062. 10.1093/database/baaa062 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Stace CA, Thompson H, Stace M: New Flora of the British Isles. C&M Floristics;2019. [ Google Scholar ] Stroh PA, Walker KJ, Humphrey TA, et al. : Plant Atlas 2020: Mapping Changes in the Distribution of the British and Irish Flora. Durham UK; Oxford UK: Botanical Society of Britain and Ireland; Princeton University Press;2023. [ Google Scholar ] Twyford AD, Beasley J, Barnes I, et al. : A DNA barcoding framework for taxonomic verification in the Darwin Tree of Life Project. Wellcome Open Res. 2024;9:339. 10.12688/wellcomeopenres.21143.1 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Vasimuddin M, Misra S, Li H, et al. : Efficient architecture-aware acceleration of BWA-MEM for multicore systems. 2019 IEEE International Parallel and Distributed Processing Symposium (IPDPS). IEEE;2019;314–24. 10.1109/IPDPS.2019.00041 [ DOI ] [ Google Scholar ] Zhou C, McCarthy SA, Durbin R: YaHS: Yet another Hi-C scaffolding tool. Bioinformatics. 2023;39(1). 10.1093/bioinformatics/btac808 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Wellcome Open Res. 2026 Apr 20. doi: 10.21956/wellcomeopenres.28759.r152433 Reviewer response for version 1 Margaret E Staton Margaret E Staton 1 The University of Tennessee, Knoxville, Tennessee, USA Referee Find articles by Margaret E Staton 1 Author information Copyright and License information 1 The University of Tennessee, Knoxville, Tennessee, USA Competing interests: No competing interests were disclosed. Roles Margaret E Staton : Referee Copyright: © 2026 Staton ME This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. PMC Copyright notice The authors present a chromosome scale haploid reference genome for Geranium rotundifolium. The genome used HiFi and HiC to yield a high quality final product. The authors might comment on why they opted to use hifiasm with the -primary flag instead of haplotype resolved assemblies, which are usually possible when hi-C data is included. Its unclear if the gene annotation utilized the RNASeq data or if the genes were functionally annotated. There's also no information on the repetitive content. The genome was annotated by the Ensembl pipeline, but some results on these points would be a welcome addition. Are sufficient details of methods and materials provided to allow replication by others? Yes Is the rationale for creating the dataset(s) clearly described? Yes Are the datasets clearly presented in a useable and accessible format? Yes Are the protocols appropriate and is the work technically sound? Yes Reviewer Expertise: Computational genomics 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. Wellcome Open Res. 2026 Apr 6. doi: 10.21956/wellcomeopenres.28759.r152432 Reviewer response for version 1 Nils Stein Nils Stein 1 Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Seeland, Germany Referee Find articles by Nils Stein 1 Author information Copyright and License information 1 Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Seeland, Germany Competing interests: No competing interests were disclosed. Roles Nils Stein : Referee Copyright: © 2026 Stein N This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. PMC Copyright notice The authors present a de novo nuclear and plastid genome assembly of G. rotundifolium. The species was selected as being endemic to UK and thus became a selected species within the DToL initiative. The study follows transparently the established standardized procedures and all assembly metrics and stats are presented and benchmarked against the Earth Biogenome Project standards. I am reviewing the first time for this format but I am absolutely thrilled - this is, to my opinion the way genome sequences should be presented and made public rapidly in order to generate impact in the science community comments: 1) HiC interaction plot: I understand that the effort is limited that can be spent in a high throughput genome sequencing effort for individual curation steps. Individual chromosomes still have obvious traces of orientation/assembly artifacts. It also looks as if chromosomes are presented in random orientation. There is a convention that chromosomes shall be presented from short to long arm. It looks as if this species has a mix of acrocentric, telocentric and metacentric chromosomes. If there are any cytological data the chromosome order could be referred to this would add value. Are sufficient details of methods and materials provided to allow replication by others? Yes Is the rationale for creating the dataset(s) clearly described? Yes Are the datasets clearly presented in a useable and accessible format? Yes Are the protocols appropriate and is the work technically sound? Yes Reviewer Expertise: Plant Genetics, Plant Genomics, Plant Genetic Resources 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. Associated Data This section collects any data citations, data availability statements, or supplementary materials included in this article. Data Availability Statement European Nucleotide Archive: G. rotundifolium (round-leaved crane’s-bill). Accession number PRJEB69504 . The genome sequence is released openly for reuse. The G. rotundifolium genome sequencing initiative is part of the Darwin Tree of Life Project (PRJEB40665) and Sanger Institute Tree of Life Programme (PRJEB43745). 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 Tables 1 and 2 . Pipelines used for genome assembly at the WSI Tree of Life are available at https://pipelines.tol.sanger.ac.uk/pipelines . Table 5 lists software versions used in this study. Table 5. Software versions and sources. Software Version Source BEDTools 2.30.0 https://github.com/arq5x/bedtools2 BLAST 2.14.0 ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/ BlobToolKit 4.3.9 https://github.com/blobtoolkit/blobtoolkit BUSCO 5.5.0 https://gitlab.com/ezlab/busco bwa-mem2 2.2.1 https://github.com/bwa-mem2/bwa-mem2 Cooler 0.8.11 https://github.com/open2c/cooler DIAMOND 2.1.8 https://github.com/bbuchfink/diamond fasta_windows 0.2.4 https://github.com/tolkit/fasta_windows FastK 1.1 https://github.com/thegenemyers/FASTK GenomeScope2.0 2.0.1 https://github.com/tbenavi1/genomescope2.0 Gfastats 1.3.6 https://github.com/vgl-hub/gfastats GoaT CLI 0.2.5 https://github.com/genomehubs/goat-cli Hifiasm 0.19.5-r587 https://github.com/chhylp123/hifiasm HiGlass 1.13.4 https://github.com/higlass/higlass MerquryFK 1.1.2 https://github.com/thegenemyers/MERQURY.FK Minimap2 2.24-r1122 https://github.com/lh3/minimap2 Oatk 0.9 https://github.com/c-zhou/oatk MultiQC 1.14; 1.17 and 1.18 https://github.com/MultiQC/MultiQC Nextflow 23.04.1 https://github.com/nextflow-io/nextflow PretextSnapshot 0.0.5 https://github.com/sanger-tol/PretextSnapshot PretextView 0.2.5 https://github.com/sanger-tol/PretextView samtools 1.19.2 https://github.com/samtools/samtools sanger-tol/ascc 0.1.0 https://github.com/sanger-tol/ascc sanger-tol/blobtoolkit 0.4.0 https://github.com/sanger-tol/blobtoolkit Seqtk 1.3 https://github.com/lh3/seqtk Singularity 3.9.0 https://github.com/sylabs/singularity TreeVal 1.2.0 https://github.com/sanger-tol/treeval YaHS 1.2a.2 https://github.com/c-zhou/yahs Open in a new tab Articles from Wellcome Open Research are provided here courtesy of The Wellcome Trust ACTIONS View on publisher site PDF (2.0 MB) Cite Collections Permalink PERMALINK Copy RESOURCES Similar articles Cited by other articles Links to NCBI Databases Cite Copy Download .nbib .nbib Format: AMA APA MLA NLM Add to Collections Create a new collection Add to an existing collection Name your collection * Choose a collection Unable to load your collection due to an error Please try again Add Cancel Follow NCBI NCBI on X (formerly known as Twitter) NCBI on Facebook NCBI on LinkedIn NCBI on GitHub NCBI RSS feed Connect with NLM NLM on X (formerly known as Twitter) NLM on Facebook NLM on YouTube National Library of Medicine 8600 Rockville Pike Bethesda, MD 20894 Web Policies FOIA HHS Vulnerability Disclosure Help Accessibility Careers NLM NIH HHS USA.gov Back to Top