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. 2026 Jul 30;11:492. [Version 1] doi: 10.12688/wellcomeopenres.27238.1

The genome sequence of Frangula alnus Mill., 1768 (Rosales: Rhamnaceae)

Vladimir Krivtsov 1,2, Zoë A Goodwin 2, Michelle Hart 2, David Bell 2, Peter M Hollingsworth 2; Royal Botanic Garden Edinburgh Genome Acquisition Lab; Plant Genome Sizing Collective; Darwin Tree of Life Barcoding 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 Consortiuma
PMCID: PMC13562949  PMID: 42732181

Abstract

We present a genome assembly of Frangula alnus (alder buckthorn; Streptophyta; Magnoliopsida; Rosales; Rhamnaceae). The assembly consists of two haplotypes with total lengths of 279.06 megabases and 281.25 megabases. Most of haplotype 1 (99.91%) is scaffolded into 10 chromosomal pseudomolecules. Most of haplotype 2 (99.91%) is scaffolded into 10 chromosomal pseudomolecules. The mitochondrial sequence has a length of 492.03 kilobases and the plastid genome assembly has a length of 161.23 kilobases. 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: Frangula alnus, alder buckthorn, genome sequence, chromosomal, Rosales

Species taxonomy

Eukaryota; Viridiplantae; Streptophyta; Streptophytina; Embryophyta; Tracheophyta; Euphyllophyta; Spermatophyta; Magnoliopsida; Mesangiospermae; eudicotyledons; Gunneridae; Pentapetalae; rosids; fabids; Rosales; Rhamnaceae; rhamnoid group; Rhamneae; Frangula; Frangula alnus Mill., 1768 (NCBI:txid106677).

Background

Frangula alnus (alder buckthorn) is a deciduous shrub or small tree that occurs across a wide range of soil types, although it tends to avoid both drought-prone and permanently waterlogged sites ( Stroh et al., 2023). It is typically found in scrub on fen peat, at the margins of raised mires, on heathlands and valley mires, and within woodland edges, scrub and hedgerows ( Stroh et al., 2023). Within Britain and Ireland, the species is locally common in England and Wales but is considerably more scattered in Scotland and Ireland ( Stace, 2019). In Northern Ireland, F. alnus is protected under the Wildlife (Northern Ireland) Order (1985) and is recognised as a Priority Species for Conservation Action ( Bradley et al., 2009). Its native range extends from Europe to Central Siberia and Xinjiang, with populations also occurring in north-west Africa ( POWO, 2026).

Frangula alnus is reported to be diploid (2 n = 20), with three chromosome counts of this number reported from British and Irish populations ( Godwin, 1943; Gornall et al., 2021; Rutland, 1941).

Alder Bbckthorn is an ecologically significant species that supports a diverse range of wildlife. Notably, it is one of only two larval host plants for the Common Brimstone butterfly ( Gonepteryx rhamni), whose distribution in Britain has been shown to closely track the natural range of its host plants ( Cowley et al., 2000; Gutiérrez & Thomas, 2000).

Landscape genomic studies of native European populations indicate that genetic variation in Frangula alnus is influenced by environmental selection, with precipitation and temperature identified as important drivers of adaptive differentiation ( De Kort et al., 2015).

The high-quality genome sequence presented here provides an important resource for investigating the genetic basis of adaptation and resilience in this species as environmental conditions continue to change. In addition, genomic resources could facilitate research into the coevolutionary relationship between F. alnus and specialist species such as the Brimstone butterfly.

The assembly was produced using the Tree of Life pipeline from a specimen collected from Royal Botanic Garden Edinburgh (Inverleith), Scotland, UK (55.97, −3.21) ( Figure 1).

Figure 1. Photographs of the Frangula alnus specimen (drFraAlnu1) from which samples were taken for genome sequencing.


Figure 1.

Methods

Sample acquisition, flow cytometry and DNA barcoding

A specimen of Frangula alnus (specimen ID EDTOL00065, ToLID drFraAlnu1; Figure 1) was used for genome sequencing. It was collected from Royal Botanic Garden Edinburgh Scotland, UK (latitude 55.9652, longitude −3.2092) on 2022-08-08. The specimen was collected and identified by Vladimir Krivtsov (Royal Botanic Garden Edinburgh). The herbarium voucher associated with the sequenced plant is has been deposited in the herbarium of RBG Edinburgh (E) https://data.rbge.org.uk/herb/E01358629.

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 Solanum lycopersicum L. ‘Stupické polní rané’ with an assumed 1C-value of 968 Mb ( Doležel et al., 2007).

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 drFraAlnu1 sample was weighed and triaged to determine the appropriate extraction protocol. For HMW DNA extraction, 94.0 mg of leaf tissue was homogenised by cryogenic bead beating. The Plant Organic Extraction protocol was used. DNA was then 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 12.1 ng/μL with a fragment size of 15.6 kb.

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 on a Revio instrument (Pacific Biosciences, California, USA). Prepared libraries selected for multiplexing were pooled based on genome size, library molarity, and the intended plex level. Primers were annealed and polymerases were bound to generate circularised complexes, following the manufacturer’s instructions. Complexes were purified using SMRTbell beads, diluted to the Revio loading concentration, and spiked with a Revio sequencing internal control. The pooled libraries were sequenced on a Revio 25 M Plex SMRT cell in a 3-plex run. SMRT Link software (Pacific Biosciences), a web-based workflow manager, was used to configure and monitor the run and to carry out primary and secondary data analysis.

Hi-C

Sample preparation and crosslinking

Hi-C data were generated from the leaf tissue of drFraAlnu1 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 in Hi-C phasing mode ( Cheng et al., 2021, 2022), producing two haplotypes. Hi-C reads ( Rao et al., 2014) were mapped to the primary contigs using bwa-mem2 ( Vasimuddin et al., 2019). Contigs were further scaffolded with Hi-C data in YaHS ( Zhou et al., 2023), using 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 OATK ( Zhou et al., 2025). The comparator for the mitochondrial assembly was the mitochondrial genome of Ziziphus jujuba (NC_029809.1).

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 29 breaks and 122 joins. This reduced the scaffold count by 71.6%, reduced the scaffold N50 by 2.3%, and increased the total assembly length by 0.8%. The curation process is described at sanger-tol/curation-resources . 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 both 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). PacBio reads were aligned to the assembly using minimap2 ( Li, 2018) and processed with SAMtools ( Danecek et al., 2021) to generate coverage tracks. The pipeline runs BUSCO ( Manni et al., 2021) using lineages identified from the NCBI Taxonomy ( Schoch et al., 2020). For the three basal BUSCO lineages, eukaryota, bacteria and archaea, BUSCO genes are aligned to the UniProt Reference Proteomes database ( Bateman et al., 2023) using DIAMOND BLASTp ( Buchfink et al., 2021). The genome assembly 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 using DIAMOND BLASTx. Sequences without DIAMOND BLASTx hits are extracted using seqtk and aligned to the NT database using BLASTn ( Altschul et al., 1990). The outputs are consolidated into a BlobDir for visualisation in BlobToolKit. 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 Frangula alnus was sequenced using Pacific Biosciences single-molecule HiFi long reads, generating 27.26 Gb (gigabases) from 2.73 million reads, which were used to assemble the genome. GenomeScope2.0 analysis using a p = 2 model gave a k-mer-based genome size estimate of 314.46 Mb, with a heterozygosity of 0.38% and repeat content of 46.70% ( Figure 2). Using flow cytometry, the genome size (1C-value) of the sample was estimated to be 0.36 pg, equivalent to 350.00 Mb. These estimates guided expectations for the assembly. Based on the estimated genome size, the sequencing data provided approximately 84× coverage. Hi-C sequencing produced 119.89 Gb from 396.97 million read pairs, 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.


Figure 2.

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 Frangula alnus (BioProject PRJEB73435).

Platform PacBio HiFi Hi-C
ToLID drFraAlnu1 drFraAlnu1
Specimen ID EDTOL00065 EDTOL00065
BioSample (source individual) SAMEA7535977 SAMEA7535977
BioSample (tissue) SAMEA111431655 SAMEA111431656
Tissue leaf leaf
Instrument Revio Illumina NovaSeq 6000
Run accessions ERR12721077 ERR12723492
Read count total 2.73 million reads 396.97 million read pairs
Base count total 27.26 Gb 119.89 Gb

Assembly statistics

The genome was assembled into two haplotypes using Hi-C phasing. Haplotype 1 was curated to chromosome level, while haplotype 2 was assembled to scaffold level. The final assembly has a total length of 279.06 Mb in 21 scaffolds, with 127 gaps, and a scaffold N50 of 24.62 Mb ( Table 2).

Table 2. Genome assembly statistics for Frangula alnus.

Genome assembly Haplotype 1 Haplotype 2
Assembly name drFraAlnu1.hap1.1 drFraAlnu1.hap2.1
Assembly accession GCA_964106965.1 GCA_964106655.1
Assembly level chromosome chromosome
Span (Mb) 279.06 281.25
Number of chromosomes 10 10
Number of contigs 148 135
Contig N50 7.34 Mb 7.04 Mb
Number of scaffolds 21 18
Scaffold N50 24.62 Mb 25.06 Mb
Longest scaffold length (Mb) 50.97 51.05
Organelles Mitochondrial genome: 492.03 kb; Plastid genome: 161.23 kb -

The scaffold count follows the NCBI assembly statistics and excludes organelle assemblies, which are reported separately where present.

Most of the assembly sequence (99.91%) 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). This genome has been assembled using PacBio and HiC data and phased. The result is two curated haplotypes. Chromosome 8 contains a large haplotypic inversion between ~17.8 and 20.5 Mbp (Hap 2 coordinates). The following regions of haplotype 1 are of uncertain order and orientation: Chromosome 2 ~ 7.3–10.9 Mbp, Chromosome 8 ~ 13.2–14.2 Mbp. The following regions of haplotype 2 are of uncertain order and orientation: Chromosome 2 ~ 7.1–9.5 Mbp, Chromosome 8 ~ 13.2–16.9 Mbp.

Figure 3. Hi-C contact map of the Frangula alnus genome assembly.


Figure 3.

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 both haplotypes of the genome assembly of Frangula alnus, drFraAlnu1.

Haplotype 1 Haplotype 2
INSDC accession Name Length (Mb) GC% INSDC accession Name Length (Mb) GC%
OZ066669.1 1 50.97 35 OZ066628.1 1 51.05 35
OZ066670.1 2 41.97 37 OZ066629.1 2 40.55 36
OZ066671.1 3 25.20 35.50 OZ066631.1 3 25.06 35.50
OZ066672.1 4 24.62 35 OZ066634.1 4 24.27 35
OZ066673.1 5 24.43 35 OZ066633.1 5 24.44 35
OZ066674.1 6 24.28 35 OZ066630.1 6 25.44 35.50
OZ066675.1 7 22.99 35 OZ066635.1 7 23.24 35
OZ066676.1 8 22.16 35.50 OZ066632.1 8 24.49 36
OZ066677.1 9 21.39 35 OZ066636.1 9 21.44 35
OZ066678.1 10 20.79 35 OZ066637.1 10 21.01 35

The mitochondrial genome (length 492.03 kb, OZ066679.1) and plastid genome (length 161.23 kb, OZ066680.1) were also assembled. These sequences are included as contigs in the multifasta file of the genome submission and as standalone records.

Assembly quality metrics

For haplotype 1, the estimated QV is 61.9, and for haplotype 2, 62.6. When the two haplotypes are combined, the assembly achieves an estimated QV of 62.2. The k-mer completeness is 89.80% for haplotype 1, 89.87% for haplotype 2, and 99.03% for the combined haplotypes ( Figure 4).

Figure 4. Evaluation of k-mer completeness using MerquryFK.


Figure 4.

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 assemblies, and the red and blue curves show k-mers found only in one assembly.

BUSCO analysis using the eudicots_odb10 reference set ( n = 2 326) identified 98.2% of the expected gene set (single = 95.1%, duplicated = 3.1%) for haplotype 1. The snail plot in Figure 5 summarises the scaffold length distribution and other assembly statistics for haplotype 1. The blob plot in Figure 6 shows the distribution of scaffolds by GC proportion and coverage for haplotype 1.

Figure 5. Assembly metrics for drFraAlnu1.hap1.1.


Figure 5.

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 drFraAlnu1.hap1.1.


Figure 6.

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 January 2026. The EBP metric calculated for haplotype 1 is 6.C.Q61, meeting the recommended 6.C.Q40 reference standard.

Table 4. Earth biogenome project summary metrics for the Frangula alnus assembly.

Measure Value Benchmark
EBP summary (haplotype 1) 6.C.Q61 6.C.Q40
Contig N50 length 7.34 Mb ≥ 1 Mb
Scaffold N50 length 24.62 Mb = chromosome N50
Consensus quality (QV) Haplotype 1: 61.9; haplotype 2: 62.6; combined: 62.2 ≥ 40
k-mer completeness Haplotype 1: 89.80%; Haplotype 2: 89.87%; combined: 99.03% ≥ 90%
BUSCO C:98.2% [S:95.1%, D:3.1%], F:0.3%, M:1.5%, n:2 326 S > 90%; D < 5%
Percentage of assembly assigned to chromosomes 99.91% ≥ 90%

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.

Author information

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: Frangula alnus (alder buckthorn). Accession number PRJEB73435; https://identifiers.org/ena.embl/PRJEB73435. The genome sequence is released openly for reuse. The Frangula alnus genome sequencing initiative is part of the Darwin Tree of Life Project (PRJEB40665) and the 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 used for Frangula alnus.

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 0.19.8-r603 https://github.com/chhylp123/hifiasm
HiGlass 1.13.4 https://github.com/higlass/higlass
MerquryFK 1.1.0-c1 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; 23.10.0 https://github.com/nextflow-io/nextflow
PretextSnapshot 0.0.4 https://github.com/sanger-tol/PretextSnapshot
PretextView 1.0.3 https://github.com/sanger-tol/PretextView
samtools 1.16.1; 1.17; 1.18; 1.19.2; 1.6 https://github.com/samtools/samtools
sanger-tol/ascc 0.1.0 https://github.com/sanger-tol/ascc
sanger-tol/blobtoolkit 0.6.0 https://github.com/sanger-tol/blobtoolkit
sanger-tol/curationpretext 1.4.2 https://github.com/sanger-tol/curationpretext
Seqtk 1.4-r122 https://github.com/lh3/seqtk
Singularity 3.9.0 https://github.com/sylabs/singularity
TreeVal 1.0.2 https://github.com/sanger-tol/treeval
YaHS 1.2a.2 https://github.com/c-zhou/yahs

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Wellcome Open Res. 2026 Sep 4. doi: 10.21956/wellcomeopenres.29979.r163251

Reviewer response for version 1

Hari Paul Sankhyan 1, Sumit Nangla 2

Summary

The manuscript presents a high-quality, chromosome-level genome assembly of Frangula alnus (alder buckthorn), generated as part of the Darwin Tree of Life project. The authors provide a phased genome assembly comprising two haplotypes, together with mitochondrial and plastid genome sequences. The study is supported by extensive specimen documentation, flow cytometry, DNA barcoding, PacBio HiFi sequencing, Hi-C scaffolding, and a well-described assembly and quality-assessment workflow.

The manuscript provides a clear biological and scientific rationale for generating a reference genome for F. alnus. The species is ecologically important, including its role as a host plant for specialist species such as the Brimstone butterfly, and genomic resources may contribute to future studies on adaptation, environmental resilience, and coevolutionary relationships. The assembly therefore represents a valuable genomic resource for plant evolutionary biology, ecology, conservation, and comparative genomics.

Overall, the manuscript is well structured, technically sound, and provides a valuable genomic dataset. The genome assembly appears to be of high quality, with most of both haplotypes assigned to chromosomal pseudomolecules and supported by multiple complementary quality assessments.

Assessment of the specific questions

1. Is the rationale for creating the dataset(s) clearly described? — Yes

The rationale for generating the Frangula alnus genome assembly is clearly explained. The manuscript provides sufficient biological and ecological background on the species and identifies the potential value of a high-quality genome sequence for investigating adaptation, resilience, genetic variation, and ecological interactions. The inclusion of this species within the Darwin Tree of Life initiative further provides a clear broader rationale for the generation of the dataset.

2. Are the protocols appropriate and is the work technically sound? — Yes

The experimental and computational approaches are appropriate for the generation of a high-quality plant reference genome. The use of PacBio HiFi sequencing in combination with Hi-C data and haplotype-aware assembly provides a robust strategy for producing phased, chromosome-level assemblies.

The workflow includes appropriate specimen identification and validation, DNA barcoding, high molecular weight DNA extraction, long-read sequencing, Hi-C library preparation, genome assembly, scaffolding, and quality assessment. The use of complementary assembly evaluation approaches, including k-mer analysis, Merqury-based quality assessment, BUSCO completeness assessment, and BlobToolKit visualisation, provides substantial support for the quality and completeness of the final assembly.

The reported quality metrics are also convincing. The manuscript reports high estimated consensus quality values and high combined k-mer completeness, while BUSCO analysis recovered 98.2% of the expected eudicot gene set for haplotype 1. The assembly therefore meets a high standard for a reference genome resource.

3. Are sufficient details of methods and materials provided to allow replication by others? — Yes

The manuscript provides substantial methodological detail covering specimen collection, taxonomic identification, herbarium voucher deposition, genome size estimation, DNA barcoding, DNA extraction, library preparation, sequencing, Hi-C generation, genome assembly, scaffolding, and quality assessment.

The authors also provide references to established protocols and standard operating procedures, including protocols for DNA extraction, sample processing, sequencing, and genome analysis. Software tools and versions are reported, which further improves reproducibility.

The description of the sequencing and assembly workflow is sufficiently detailed to enable researchers to understand and reproduce the general analytical approach, particularly when used in conjunction with the cited protocols and software documentation.

4. Are the datasets clearly presented in a useable and accessible format? — Yes

The datasets are clearly presented through tables, figures, accession information, and genome assembly statistics. The manuscript provides information on the specimen and sequencing dataset, assembly statistics, chromosomal pseudomolecules, organellar genome assemblies, and assembly quality metrics.

The presentation of the two haplotypes is particularly useful, and the use of Hi-C contact maps, k-mer spectra, Merqury assessment, BUSCO analysis, and BlobToolKit plots provides multiple complementary perspectives on assembly quality. The manuscript also clearly reports regions where scaffold order and orientation remain uncertain, which is an important and transparent aspect of genome resource reporting.

Overall assessment

This manuscript presents a valuable and high-quality genomic resource for Frangula alnus. The rationale for producing the dataset is clearly articulated, the experimental and computational protocols are appropriate and technically sound, and the methods are described with sufficient detail to support reproducibility. The genome assembly is supported by comprehensive quality-control analyses and appears to meet a high standard for a chromosome-level reference genome.

The manuscript is clearly written and appropriately structured for a genome resource publication. I did not identify any major scientific concerns that would compromise the validity or utility of the dataset.

Recommendation

All four assessment questions: Yes.

In my opinion, the manuscript is scientifically sound and the dataset represents a useful contribution to genomic resources for Frangula alnus and related research areas. I recommend indexing in its present form, subject to any routine editorial corrections.

Are sufficient details of methods and materials provided to allow replication by others?

Yes

Is the rationale for creating the dataset(s) clearly described?

No

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:

Forestry; Plant Genetics and Breeding; Tree Improvement; Plant Genomics; Forest Biotechnology; Biodiversity and Conservation Genetics

We confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Wellcome Open Res. 2026 Aug 6. doi: 10.21956/wellcomeopenres.29979.r163249

Reviewer response for version 1

S Brooks Parrish 1

This manuscript presents a high-quality, chromosome-scale, haplotype-resolved genome assembly for  Frangula alnus, alder buckthorn. The rationale for generating this dataset is clearly described. F. alnus is an ecologically important and conservation-relevant species, including its role as a host plant for the Common Brimstone butterfly, and the genome will support future studies of adaptation, resilience, and species interactions. The assembly consists of two haplotypes of 279.06 Mb and 281.25 Mb, with 99.91% of each haplotype scaffolded into 10 chromosomal pseudomolecules. Mitochondrial and plastid assemblies are also reported.

The protocols are appropriate and the work is technically sound. The authors used suitable procedures for sample collection, species verification by DNA barcoding, genome size estimation by flow cytometry and k-mer analysis, PacBio HiFi sequencing, Hi-C scaffolding, assembly, manual curation, and quality assessment. The reported metrics indicate an excellent assembly, including scaffold N50 values of 24.62 Mb and 25.06 Mb, high consensus quality values, combined k-mer completeness of 99.03%, and BUSCO completeness of 98.2%. Sufficient methodological detail, software versions, specimen information, and accession numbers are provided to support reproducibility.

The datasets are clearly presented in usable and accessible formats, with raw sequence data and assemblies deposited in INSDC databases. I have no major concerns and no points that must be addressed to make the article scientifically sound. I recommend approval. As minor editorial suggestions, the authors may want to clarify the difference between the flow-cytometry genome size estimate and final assembly, comment on the practical implications of the noted uncertain chromosome regions and chromosome 8 inversion, and correct the typo “Alder Bbckthorn.”

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 Breeding, Bioinformatics, Molecular Biology

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Data Availability Statement

    European Nucleotide Archive: Frangula alnus (alder buckthorn). Accession number PRJEB73435; https://identifiers.org/ena.embl/PRJEB73435. The genome sequence is released openly for reuse. The Frangula alnus genome sequencing initiative is part of the Darwin Tree of Life Project (PRJEB40665) and the 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 used for Frangula alnus.

    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 0.19.8-r603 https://github.com/chhylp123/hifiasm
    HiGlass 1.13.4 https://github.com/higlass/higlass
    MerquryFK 1.1.0-c1 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; 23.10.0 https://github.com/nextflow-io/nextflow
    PretextSnapshot 0.0.4 https://github.com/sanger-tol/PretextSnapshot
    PretextView 1.0.3 https://github.com/sanger-tol/PretextView
    samtools 1.16.1; 1.17; 1.18; 1.19.2; 1.6 https://github.com/samtools/samtools
    sanger-tol/ascc 0.1.0 https://github.com/sanger-tol/ascc
    sanger-tol/blobtoolkit 0.6.0 https://github.com/sanger-tol/blobtoolkit
    sanger-tol/curationpretext 1.4.2 https://github.com/sanger-tol/curationpretext
    Seqtk 1.4-r122 https://github.com/lh3/seqtk
    Singularity 3.9.0 https://github.com/sylabs/singularity
    TreeVal 1.0.2 https://github.com/sanger-tol/treeval
    YaHS 1.2a.2 https://github.com/c-zhou/yahs

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