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[Version 1] doi: 10.12688/wellcomeopenres.26151.1 Search in PMC Search in PubMed View in NLM Catalog Add to search The genome sequence of the sweet violet, Viola odorata L. (Malpighiales: Violaceae) 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, Writing – Original Draft Preparation, Writing – Review & Editing Find articles by Maarten J M Christenhusz 1, 2 , Michael F Fay Michael F Fay 1 Royal Botanic Gardens Kew, Richmond, England, UK 3 The University of Western Australia, Crawley, Western Australia, Australia Writing – Review & Editing Find articles by Michael F Fay 1, 3 , Ilia J Leitch Ilia J Leitch 1 Royal Botanic Gardens Kew, Richmond, England, UK Writing – Review & Editing Find articles by Ilia J Leitch 1 ; 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 The University of Western Australia, Crawley, Western Australia, Australia a Email: [email protected] No competing interests were disclosed. Roles Maarten J M Christenhusz : Investigation, Resources, Writing – Original Draft Preparation, Writing – Review & Editing Michael F Fay : Writing – Review & Editing Ilia J Leitch : Writing – Review & Editing Accepted 2026 Mar 3; 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: PMC13058574 PMID: 41960579 Abstract We present a genome assembly of Viola odorata (Sweet violet; Streptophyta; Magnoliopsida; Malpighiales; Violaceae). The genome sequence has a total length of 698.62 megabases. Most of the assembly (99.9%) is scaffolded into 10 chromosomal pseudomolecules. The mitochondrial sequence has a length of 482.11 kilobases and the plastid genome assembly has a length of 158.28 kilobases. Gene annotation of this assembly on Ensembl identified 34 902 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: Viola odorata; Sweet violet; genome sequence; chromosomal; Malpighiales Species taxonomy Eukaryota; Viridiplantae; Streptophyta; Streptophytina; Embryophyta; Tracheophyta; Euphyllophyta; Spermatophyta; Magnoliopsida; Mesangiospermae; eudicotyledons; Gunneridae; Pentapetalae; rosids; fabids; Malpighiales; Violaceae; Viola ; Viola subgen. Viola ; Viola sect. Viola ; Viola subsect. Viola ; Viola odorata L. (NCBI:txid97441). Background Sweet violet, V. odorata (Violaceae) is a perennial, creeping herb with heart-shaped leaves, and in early spring, flowers with violet, purple or white petals. The flowers are spurred and highly scented, and pollinated by solitary bees. Cleistogamous, self-pollinating flowers are also often formed. Plants usually grow in shaded places, under hedges, in woodland margins or in clearings. It is widespread across Europe, western Asia and Northwest Africa, and is naturalised in the Nordic countries, North and South America, South and East Asia, Australia and New Zealand ( POWO, 2026 ). It can be found in most of Britain and Ireland, but it is very local in Scotland, Ireland, West Wales and the Channel Islands. Due to its popularity, some populations may be the result of garden escapes ( Stace, 2019 ). Because of its early flowering and sweet and attractive scent, special powers have been attributed to sweet violets since ancient times. In Greek mythology, the flower is known as ion porfuroun , and it was the symbol of Persephone, the goddess of the underworld, because it flowered so early that Persephone would not yet have emerged from the nether realm. Its violet flowers were a symbol of purity and virginity, of faithfulness and durability, and a sign of perpetual love. In the latter half of the 19th century it became popular for men to wear small bouquets of violets. For instance, Oscar Wilde is depicted wearing a small posy on a string attached to his belt ( De Cleene & Lejeune, 2000 ). As an emblem, V. odorata was also chosen for the imperial Napoleonic party (1769–1821), but during the Restoration this symbol was outlawed ( De Cleene & Lejeune, 2000 ). V. odorata is probably the most economically important species of violet ( Wyse Jackson, 2014 ). It has been used to make perfume for over 2000 years, and it is grown commercially in southern France for the production of the essential oils for this purpose. It is also used as a skin cleanser and in soaps and creams, not only for its scent and colour, but also for its medicinal properties. Extracts of violets are used in syrups, liqueurs, confectionery and cakes, especially in England and France ( De Cleene & Lejeune, 2000 ). Fothergill (1944) reported a diploid complement of 2 n = 20 across several named varieties of V. odorata , with a karyotype formula of 2(B, 9C) based on four size classes (A very long, B long, C medium, D small). He noted that the chromosomes are generally similar to those of V. hirta , but that V. odorata consistently includes a pair of long B-type chromosomes that are absent in V. hirta , whereas V. hirta has a pair of D-type chromosomes absent in V. odorata. 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, we sequenced the genome of the sweet violet, V. odorata L. Here we present a chromosome-level genome sequence based on a wild specimen from Petersham Common, Surrey, UK ( Figure 1 ). Figure 1. Photograph of the Viola odorata (ddVioOdor1) specimen from which samples were taken for genome sequencing. Open in a new tab Methods Sample acquisition, flow cytometry and DNA barcoding A specimen of V. odorata (specimen ID KDTOL10129, ToLID ddVioOdor1; Figure 1 ) was used for genome sequencing. It was collected from Richmond, Surrey, United Kingdom (latitude 51.4472, longitude −0.298) on 2021-03-09. The specimen was collected and identified by Maarten J. M. Christenhusz (Royal Botanic Gardens Kew). The herbarium voucher associated with the sequenced plant is K001400828 and is deposited in the herbarium of RBG Kew (K). 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. CyStain™ PI OxProtect Staining Buffer (Sysmex UK Ltd) 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 ). 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 ddVioOdor1 sample was weighed and triaged to determine the appropriate extraction protocol. Leaf tissue was homogenised by cryogenic disruption using the Covaris cryoPREP® Automated Dry Pulverizer. Two different extractions of HMW were performed, one using the Automated Plant MagAttract v2 protocol, and the other using the Plant Organic Extraction protocol. DNA was sheared into an average fragment size of 12–20 kb following the Megaruptor ® 3 for LI PacBio protocol. 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 17.1 ng/μL and a yield of 2 223.00 ng. PacBio HiFi library preparation and sequencing Library preparation and sequencing were performed at the WSI Scientific Operations core. Libraries were prepared using the SMRTbell Prep Kit 3.0 (Pacific Biosciences) according to the manufacturer’s instructions. The kit includes reagents for end repair/A-tailing, adapter ligation, post-ligation SMRTbell bead clean-up, and nuclease treatment. Size selection and clean-up were performed using diluted AMPure PB beads (Pacific Biosciences). DNA concentration was quantified using a Qubit Fluorometer v4.0 (ThermoFisher Scientific) and the Qubit 1X dsDNA HS assay kit. Final library fragment size was assessed with the Agilent Femto Pulse Automated Pulsed Field CE Instrument (Agilent Technologies) using the gDNA 55 kb BAC analysis kit. 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 ddVioOdor1 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. 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 MerquryFK ( Rhie et al. , 2020 ). The organelle genomes were assembled using MitoHiFi ( Uliano-Silva et al. , 2023 ) and OATK ( Zhou et al. , 2025 ). 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 17 breaks and 95 joins. This reduced the scaffold count by 73.8% and increased the scaffold N50 by 6.7%. The curation process is described 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 MerquryFK 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 database ( 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 by querying NCBI datasets ( O’Leary et al. , 2024 ). 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 containerisation through Docker ( Merkel, 2014 ) and Singularity ( Kurtzer et al. , 2017 ). Genome sequence report Sequence data The genome of a specimen of V. odorata was sequenced using Pacific Biosciences single-molecule HiFi long reads, generating 22.61 Gb (gigabases) from 1.84 million reads, which were used to assemble the genome. GenomeScope2.0 analysis estimated the haploid genome size at 371.80 Mb, with a heterozygosity of 7.22% and repeat content of 54.83% ( Figure 2 ). Using flow cytometry, the genome size (1C-value) of the sample was estimated to be 1.05 pg, equivalent to 1 030.00 Mb. These estimates guided expectations for the assembly. Based on the estimated genome size, the sequencing data provided approximately 57× coverage. Hi-C sequencing produced 104.71 Gb from 693.43 million reads, which were used to scaffold the assembly. 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 PRJEB69496. Platform PacBio HiFi Hi-C ToLID ddVioOdor1 ddVioOdor1 Specimen ID KDTOL10129 KDTOL10129 BioSample (source individual) SAMEA9143020 SAMEA9143020 BioSample (tissue) SAMEA9143630 SAMEA9143629 Tissue leaf leaf Instrument Sequel IIe Illumina NovaSeq 6000 Run accessions ERR12303932 ERR12318578 Read count total 1.84 million 693.43 million Base count total 22.61 Gb 104.71 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 698.62 Mb in 25 scaffolds, with 484 gaps, and a scaffold N50 of 70.51 Mb ( Table 2 ). Table 2. Genome assembly statistics. Assembly name ddVioOdor1.1 Assembly accession GCA_963691705.1 Alternate haplotype accession GCA_963691845.1 Assembly level chromosome Span (Mb) 698.62 Number of chromosomes 10 Number of contigs 509 Contig N50 2.78 Mb Number of scaffolds 25 Scaffold N50 70.51 Mb Organelles Mitochondrion: 482.11 kb; Plastid: 158.28 kb Open in a new tab Most of the assembly sequence (99.9%) was assigned to 10 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 Viola odorata 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 Viola odorata ddVioOdor1. INSDC accession Molecule Length (Mb) GC% OY829537.1 1 86.17 40.50 OY829538.1 2 82.27 40 OY829539.1 3 76.95 40 OY829540.1 4 73.59 40 OY829541.1 5 70.51 40.50 OY829542.1 6 66.10 39.50 OY829543.1 7 65.05 40 OY829544.1 8 60.60 39.50 OY829545.1 9 60.34 40 OY829546.1 10 56.31 39.50 Open in a new tab The mitochondrial genome (length 482.11 kb, OY829547.1 ) and plastid genome (length 158.28 kb, OY829548.1 ) 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 59.0. The k -mer completeness is 98.43% for the primary assembly, 1.30% for the alternate haplotype, and 98.44% 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 eukaryota_odb10 reference set ( n = 255) identified 99.2% of the expected gene set (single = 13.3%, duplicated = 85.9%). 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 ddVioOdor1.1. 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 eukaryota_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 ddVioOdor1.1. 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.Q59 , meeting the recommended reference standard. Table 4. Earth Biogenome Project summary metrics for the Viola odorata assembly. Measure Value Benchmark EBP summary (primary) 6.C.Q59 6.C.Q40 Contig N50 length 2.78 Mb ≥ 1 Mb Scaffold N50 length 70.51 Mb = chromosome N50 Consensus quality (QV) Primary: 59.3; alternate: 51.8; combined: 59.0 ≥ 40 k -mer completeness Primary: 98.43%; alternate: 1.30%; combined: 98.44% ≥ 95% BUSCO C:99.2% [S:13.3%, D:85.9%], F:0.0%, M:0.8%, n:255 S > 90%; D < 5% Percentage of assembly assigned to chromosomes 99.90% ≥ 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 V. odorata genome assembly (GCA_963691705.1) was annotated by Ensembl at the European Bioinformatics Institute (EBI). This annotation includes 59 356 transcribed mRNAs from 34 902 protein-coding and 14 061 non-coding genes. The average transcript length is 2 175.49 bp, with an average of 1.21 coding transcripts per gene and 4.51 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). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. [version 1; peer review: 3 approved] Data availability European Nucleotide Archive: V. odorata (sweet violet). Accession number PRJEB69496 . The genome sequence is released openly for reuse. The V. odorata 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. 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 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 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 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 MitoHiFi 3 https://github.com/marcelauliano/MitoHiFi 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 1.0.3 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 sanger-tol/curationpretext 1.4.2 https://github.com/sanger-tol/curationpretext Seqtk 1.3 https://github.com/lh3/seqtk Singularity 3.9.0 https://github.com/sylabs/singularity TreeVal 1.4.0 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. 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Bioinformatics. 2023;39(1). 10.1093/bioinformatics/btac808 [ DOI ] [ Google Scholar ] Wellcome Open Res. 2026 Jun 2. doi: 10.21956/wellcomeopenres.28798.r151785 Reviewer response for version 1 Zoé Postel Zoé Postel 1 Department of Ecology, Environment and Plant sciences, Stockholm University, Stockholm, Sweden Referee Find articles by Zoé Postel 1 Author information Copyright and License information 1 Department of Ecology, Environment and Plant sciences, Stockholm University, Stockholm, Sweden Competing interests: No competing interests were disclosed. Roles Zoé Postel : Referee Copyright: © 2026 Postel Z 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 This report by Christenhusz et al. describes the genome assembly of Viola odorata (Malpighiales) based on an individual sampled in Surrey, UK. The motivation, methodology, and assessment of assembly quality are clearly presented and easy to follow. Overall, the manuscript is well written, and the level of detail provided should allow future users to readily assess the quality and usability of the genome resource. The assembly itself appears to be of high structural quality, with excellent BUSCO completeness and a very high proportion of the assembly anchored to chromosome-level scaffolds. This represents a valuable resource for future research in plant biology and genomics! Additional details on the annotation process would be beneficial, although the authors appropriately refer to established protocols. Overall, this work provides a useful and well-documented genomic resource. Some more specific comments can be find below: p. 5 - In the Methods (genome assembly section), k-mer counts were performed on filtered reads. It would be helpful to clarify how these reads were filtered and which criteria were applied. p. 5 - For mitochondrial and chloroplast genome assembly, the authors used two different tools (MitoHiFi and OATK). Could the authors clarify the rationale for this choice, given that OATK can be applied to both organellar genomes? Figure 2 would benefit from additional explanation to help a broader audience interpret the different peaks. Figure 6 : a bit more help would be appreciated for interpretation from the reader p. 11 - The high proportion of duplicated BUSCOs would merit further discussion. Given the reported level of heterozygosity (7.22%) and very low k-mer completeness for the alternate assembly (1.3%), this may reflect incomplete collapsing of heterozygous regions in the primary assembly. p. 11 - Among the 34,902 protein-coding genes, it would be informative to specify how many are of mitochondrial or chloroplast origin. 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 geneticist and population genomics, nuclear and organellar genomes 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 7. doi: 10.21956/wellcomeopenres.28798.r151135 Reviewer response for version 1 Stalin Nithaniyal Stalin Nithaniyal 1 Botanical Survey of India, Western Regional Centre, Pune, Maharashtra, India Referee Find articles by Stalin Nithaniyal 1 Author information Copyright and License information 1 Botanical Survey of India, Western Regional Centre, Pune, Maharashtra, India Competing interests: No competing interests were disclosed. Roles Stalin Nithaniyal : Referee Copyright: © 2026 Nithaniyal S 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 Christenhusz and co-authors investigated genome sequence of sweet violet ( Viola odorata ) using Illumina NovaSeq 6000 as a part of Darwin Tree of Life project. This timely study focused on the notable plant V. odorata , sampled from Richmond, Surrey, United Kingdom, which is highly regarded for its cultural, horticultural, and medicinal value. The research was well-executed, utilizing a robust methodology covering DNA extraction, library preparation, genome assembly, and curation. The genome assembly represents a high-quality reference data, indicated by its high contiguity (99.9% chromosomal scaffolding) and high accuracy (QV 59.0). Furthermore, the EBP 6.C.Q59 metric demonstrates that it meets the highest international standards for reference-quality genomes. This assembly provides a strong foundation for evolutionary studies, boasting 99.2% gene-space completeness and a comprehensive Ensembl annotation of 34,902 protein-coding genes from mitochondrial, and plastid genomes. While the high gene duplication rate (85.9%) in the BUSCO analysis suggests a need for careful interpretation to distinguish between biological paralogs and potential assembly redundancies, the 99.2% overall BUSCO score confirms that the assembly captures nearly the entire expected gene set. Overall, the work is well-presented, and the genomic data will serve as a valuable resource for botanists and plant geneticists by facilitating future comparative studies. Major outcomes of the study are, 1. Achieving 99.9% of the 698.62 Mb assembly and scaffolded into 10 chromosomal pseudomolecules is a significant result and provide near-complete physical map of the genome. 2. High QV of 59.0 demonstrates quality base-call accuracy required for the reference genomes 3. Successful assembly of mitochondrial (482.11 kb) and plastid (158.28 kb) genomes 4. Ensembl Annotation provides well-curated set of 34,902 protein-coding genes which act as valuable resource to the research community Some minor comments includes, 1. Clarify the reason for high duplication rate in BUSCO analysis (85.9%) and selection of eukaryota_odb10 reference set instead of plant reference 2. A ratio of 1.2 transcripts per gene is considered low in the context of plant transcriptomics 3. Breakdown of 14 061 non-coding genes may be provided. 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: Molecular Biology, Plant Systematics, Ethnobotany 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 3. doi: 10.21956/wellcomeopenres.28798.r151782 Reviewer response for version 1 Bruno Contreras-Moreira Bruno Contreras-Moreira 1 Estacion Experimental de Aula Dei-CSIC (Ringgold ID: 54439), Zaragoza, Aragon, Spain Referee Find articles by Bruno Contreras-Moreira 1 Author information Copyright and License information 1 Estacion Experimental de Aula Dei-CSIC (Ringgold ID: 54439), Zaragoza, Aragon, Spain Competing interests: No competing interests were disclosed. Roles Bruno Contreras-Moreira : Referee Copyright: © 2026 Contreras-Moreira B 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 manuscript by Christenhusz et al describes their work on the assembly and annotation of the genome of dicot Viola odorata out of DNA extracted from a plant in Surrey. I find that the methods describe with sufficient precision all the steps from plant identification in a public park to the preparation of DNA libraries, assembly and QC. Probably the part that is explained with least detail is the gene annotation protocol, despite the info available externally. All software version/releases are also listed on Table 5. Regarding the results, the paper reports efficiently what most readers will want to know about an assembly, its quality, and its annotation, linking back to the data sources and INSDC. In addition, the genome is made available at the Ensembl browser so that anyone can use it. Here are some comments in case the authors want to address them: Figure 2 legend: Please help readers understand the figure; as it stands some people won't be able to follow what the peaks are. p8: Please provide details of the singularity container for reproducibility. p11 "Assembly quality metrics": a) I see the primary hifiasm assembly is reported; did the authors ever try a hap1/hap2 assembly or that pathway is deemed too complidated? b) why is the BUSCO completeness reported here relate to lineage eukaryota_odb10 instead of eudicots_odb1o (Figure 5)? Please explain to avoid confusion. p11 "Genome annotation report": did the gene annotation of this genome used any RNAseq data from related species or was instead based on de novo or protein-based gene calling? Are sufficient details of methods and materials provided to allow replication by others? Partly 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: Bioinformatics, plant 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. Associated Data This section collects any data citations, data availability statements, or supplementary materials included in this article. Data Availability Statement European Nucleotide Archive: V. odorata (sweet violet). Accession number PRJEB69496 . The genome sequence is released openly for reuse. The V. odorata 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. 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 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 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 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 MitoHiFi 3 https://github.com/marcelauliano/MitoHiFi 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 1.0.3 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 sanger-tol/curationpretext 1.4.2 https://github.com/sanger-tol/curationpretext Seqtk 1.3 https://github.com/lh3/seqtk Singularity 3.9.0 https://github.com/sylabs/singularity TreeVal 1.4.0 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 (2.6 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