
==== Front
Wellcome Open Res
Wellcome Open Res
Wellcome Open Research
2398-502X
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10.12688/wellcomeopenres.21191.1
Data Note
Articles
The genome sequence of lesser trefoil or Irish shamrock, Trifolium dubium Sibth. (Fabaceae)
[version 1; peer review: 2 approved]

Ruhsam Markus Investigation Resources https://orcid.org/0000-0002-8457-345X
1
Hollingsworth Peter M Investigation Resources Writing – Review & Editing https://orcid.org/0000-0003-0602-0654
1
Mc Cartney Ann M. Investigation Resources Writing – Review & Editing 2
Herron Katie E. Investigation Resources Writing – Review & Editing https://orcid.org/0009-0004-0021-5113
3
Hughes Graham M. Investigation Resources Writing – Review & Editing https://orcid.org/0000-0003-3088-345X
3
Christenhusz Maarten J. M. Writing – Review & Editing https://orcid.org/0000-0003-1398-8743
45
Fay Michael F. Writing – Original Draft Preparation https://orcid.org/0000-0003-3491-9093
4
Leitch Ilia J. Writing – Original Draft Preparation https://orcid.org/0000-0002-3837-8186
4
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
1 Royal Botanic Garden Edinburgh, Edinburgh, Scotland, UK
2 University of California Santa Cruz, Santa Cruz, California, USA
3 University College Dublin, Dublin, Leinster, Ireland
4 Royal Botanic Gardens Kew, Richmond, England, UK
5 Curtin University, Perth, Western Australia, Australia
a mark.blaxter@sanger.ac.uk
No competing interests were disclosed.

24 4 2024
2024
9 2296 3 2024
Copyright: © 2024 Ruhsam M et al.
2024
https://creativecommons.org/licenses/by/4.0/ 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.

We present a genome assembly from an individual Trifolium dubium (lesser trefoil; Tracheophyta; Magnoliopsida; Fabales; Fabaceae) as part of a collaboration between the Darwin Tree of Life and the European Reference Genome Atlas. The genome sequence is 679.1 megabases in span. Most of the assembly is scaffolded into 15 chromosomal pseudomolecules. The two mitochondrial genomes have lengths of 133.86 kb and 182.32 kb, and the plastid genome assembly has a length of 126.22 kilobases.

Cuirimid i láthair geanóm tionól ó an dhuine aonair seamróg na hÉireann mar chuid de a comhoibriú idir an Darwin Crann na Beatha agus an Atlas Géanóm Tagartha na hEorpa. Is an geanóm sheicheamhú 679.1megabases fad. An chuid is mó den tionól scafall isteach 15 crómasómach pseudomolecules. Ta an dhá mitochondrial geanóm tá faid de 133.86 kb agus 182.32 kb, agus an plastid geanóm tionól tá fad de 126.22 kb.

Trifolium dubium
lesser trefoil
genome sequence
chromosomal
Fabales
Wellcome Trust206194 218328 This work was supported by Wellcome through core funding to the Wellcome Sanger Institute [206194, <a href=https://doi.org/10.35802/206194>https://doi.org/10.35802/206194</a>] 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.
==== Body
pmcSpecies taxonomy

Eukaryota; Viridiplantae; Streptophyta; Streptophytina; Embryophyta; Tracheophyta; Euphyllophyta; Spermatophyta; Magnoliopsida; Mesangiospermae; eudicotyledons; Gunneridae; Pentapetalae; rosids; fabids; Fabales; Fabaceae; Papilionoideae; 50 kb inversion clade; NPAAA clade; Hologalegina; IRL clade; Trifolieae; Trifolium; Trifolium dubium Sibth. (NCBI:txid97021).

Background

Lesser trefoil ( Trifolium dubium Sibth.), also known as lesser hop clover or suckling clover, is a common clover species that is considered by most to represent the traditional Irish shamrock. It is native and common across Europe, north to Scandinavia and south to Morocco and Turkey, but it is also found in many temperate regions of the world as an introduced species ( POWO, 2023).

Trifolium dubium is a mat-forming annual, which has up to 20 tiny yellow flowers packed in dense globular flower heads ( Figure 1). Most commonly, it occurs in unimproved grassland, but is also found in other habitats such as lawns, pastures, coastal meadows, roadsides, waste places and disturbed areas. Its adaptability to different environmental conditions has contributed to its prevalence in both natural and anthropogenic landscapes across its range.

Figure 1. Photographs of Trifolium dubium ( a and b are representative images for the species, but not the specimen pr population used for genome sequencing, c is a representative plant from the population that was used for genome sequencing). a) https://commons.wikimedia.org/wiki/User:Rasbak b) https://commons.wikimedia.org/wiki/User:Kenraiz c) Markus Ruhsam.

There has been much discussion on the identity of the “true” shamrock, but for over a century the majority of people surveyed consider T. dubium to be the real one ( Colgan, 1892; Colgan, 1893; Nelson, 1991). Shamrock flowers from May to October in Ireland, so it is not generally in flower on St Patrick’s Day (17 March); however, leaves of T. dubium are worn on St. Patrick’s Day, and have since become a floral symbol of Ireland. Trifolim dubium appears in numerous emblems of state and non-state organisations and companies across the Republic of Ireland, Northern Ireland, and beyond. Together with the harp, the shamrock is registered as an international trademark by the Government of Ireland.

The legend of the shamrock holds that St Patrick used its three-parted clover leaflets to explain to the Irish people the Christian concept of the Holy Trinity ( Van Treeck & Croft, 1936), although the word “shamrock” derives from the Irish words seamair (clover) and óg (young) ( Nelson, 1991).

While T. dubium is not typically cultivated as a primary crop, like most legumes it is capable of fixing atmospheric nitrogen through its symbiotic relationship with nitrogen-fixing bacteria in root nodules ( Brock, 1973). This enriches the soil as well as the plants themselves, which therefore provide a good source of macro- and micronutrients and protein for livestock ( Brock, 1973; Gounden et al., 2018). This species and several related species of Trifolium also produce condensed tannins (unlike the major crop clover species T. repens L. and T. pratense L.), making them of interest to breeding programmes of forage legumes, because they are less likely to cause legume bloat in ruminants ( Fay & Dale, 1993).

While many cytological studies of Trifolium species have indicated that most (about 80%) are diploid based on x = 8 (with descending dysploidy giving rise to x = 7, 6 or 5 in some species; Ellison et al., 2006), counts of T. dubium have suggested it is a tetraploid, although there has been some discrepancy as to whether it is 2 n = 28 or 30 ( Ansari et al., 2008; Taylor et al., 1983; Vižintin et al., 2006; Zohary & Heller, 1984), or 2 n = 32 (based on a chromosome count of a plant from Kent, England; Gornall and Bailey, 1993). Recent molecular cytogenetic studies of T. dubium with 2 n = 30, are in agreement with the genome assembly reported here, and have provided important insights into its genetic composition and evolution (e.g. Ansari et al., 2008; Vozárová et al., 2021). Such studies have proposed that the species is an allotetraploid that likely arose from natural hybridisation between T. campestre Schreb. (2 n = 14) and T. micranthum Viv. (2 n = 16) ( Ansari et al., 2008).

Whole genome sequence data are now available for at least six Trifolium species (e.g. Bickhart et al., 2022; Garg et al., 2022; Griffiths et al., 2019; Santangelo et al., 2023), and here we present the first high-quality genome for T. dubium, stemming from a collaboration involving the Darwin Tree of Life Project and the European Reference Genome Atlas pilot project. We anticipate this genome will be a valuable genomic resource for a range of future studies. These include comparative analyses focused on the evolution of allopolyploid genomes, as well as studies exploring its potential as an additional nutritional source for livestock, especially given its high condensed tannin content.

Genome sequence report

The genome was sequenced from a specimen of Trifolium dubium collected from Gorebridge, UK (55.84, –3.04). Using flow cytometry, the genome size (1C-value) was estimated to be 0.84 pg, equivalent to 820 Mb. A total of 72-fold coverage in Pacific Biosciences single-molecule HiFi long reads was generated. Primary assembly contigs were scaffolded with chromosome conformation Hi-C data. Manual assembly curation corrected 283 missing joins or mis-joins, reducing the scaffold number by 61.95%, and increasing the scaffold N50 by 14.41%.

The final assembly has a total length of 679.1 Mb in 153 sequence scaffolds with a scaffold N50 of 46.0 Mb ( Table 1). The snail plot in Figure 2 provides a summary of the assembly statistics, while the distribution of assembly scaffolds on GC proportion and coverage is shown in Figure 3. The cumulative assembly plot in Figure 4 shows curves for subsets of scaffolds assigned to different phyla. Most (99.51%) of the assembly sequence was assigned to 15 chromosomal-level scaffolds. Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size ( Figure 5; Table 2). The order and orientation of contigs on chromosome 1 between 37.5 Mb and 42.4 Mb is uncertain. While not fully phased, the assembly deposited is of one haplotype. Contigs corresponding to the second haplotype have also been deposited. The mitochondrial and plastid genomes were also assembled and can be found as contigs within the multifasta file of the genome submission.

Table 1. Genome data for Trifolium dubium, drTriDubi3.1.

Project accession data	
Assembly identifier	drTriDubi3.1	
Species	Trifolium dubium	
Specimen	drTriDubi3	
NCBI taxonomy ID	97021	
BioProject	PRJEB59394	
BioSample ID	SAMEA10983579	
Isolate information	drTriDubi3: flower and leaf (DNA sequencing)
drTriDubi2: flower and leaf (Hi-C sequencing)
drTriDubi4: flower and leaf (RNA sequencing)	
Assembly metrics *	Benchmark	
Consensus quality (QV)	67.2	≥ 50	
k-mer completeness	100.0%	≥ 95%	
BUSCO **	C:98.9%[S:5.4%, D:93.6%],
F:0.1%,M:1.0%,n:5,366	C ≥ 95%	
Percentage of assembly
mapped to chromosomes	99.51%	≥ 95%	
Sex chromosomes	None	localised homologous pairs	
Organelles	Mitochondrial genomes:
133.86 kb and 182.32 kb
Plastid genome: 126.22 kb	complete single alleles	
Raw data accessions	
PacificBiosciences SEQUEL II	ERR10841331	
Hi-C Illumina	ERR10851537	
PolyA RNA-Seq Illumina	ERR10908617	
Genome assembly	
Assembly accession	GCA_951804385.1	
Accession of alternate haplotype	GCA_951804395.1	
Span (Mb)	679.1	
Number of contigs	742	
Contig N50 length (Mb)	3.0	
Number of scaffolds	153	
Scaffold N50 length (Mb)	46.0	
Longest scaffold (Mb)	64.64	
* Assembly metric benchmarks are adapted from column VGP-2020 of “Table 1: Proposed standards and metrics for defining genome assembly quality” from ( Rhie et al., 2021).

** BUSCO scores based on the fabales_odb10 BUSCO set using version 5.3.2. C = complete [S = single copy, D = duplicated], F = fragmented, M = missing, n = number of orthologues in comparison. A full set of BUSCO scores is available at https://blobtoolkit.genomehubs.org/view/drTriDubi3_1/dataset/drTriDubi3_1/busco.

Figure 2. Genome assembly of Trifolium dubium, drTriDubi3.1: metrics.

The BlobToolKit Snailplot shows N50 metrics and BUSCO gene completeness. The main plot is divided into 1,000 size-ordered bins around the circumference with each bin representing 0.1% of the 679,499,717 bp assembly. The distribution of scaffold lengths is shown in dark grey with the plot radius scaled to the longest scaffold present in the assembly (64,644,275 bp, shown in red). Orange and pale-orange arcs show the N50 and N90 scaffold lengths (46,006,535 and 34,190,264 bp), respectively. The pale grey spiral shows the cumulative scaffold count on a log scale with white scale lines showing successive orders of magnitude. The blue and pale-blue area around the outside of the plot shows the distribution of GC, AT and N percentages in the same bins as the inner plot. A summary of complete, fragmented, duplicated and missing BUSCO genes in the fabales_odb10 set is shown in the top right. An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/drTriDubi3_1/dataset/drTriDubi3_1/snail.

Figure 3. Genome assembly of Trifolium dubium, drTriDubi3.1: BlobToolKit GC-coverage plot.

Scaffolds are coloured by phylum. Circles are sized in proportion to scaffold length. Histograms show the distribution of scaffold length sum along each axis. An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/drTriDubi3_1/dataset/drTriDubi3_1/blob.

Figure 4. Genome assembly of Trifolium dubium, drTriDubi3.1: BlobToolKit cumulative sequence plot.

The grey line shows cumulative length for all scaffolds. Coloured lines show cumulative lengths of scaffolds assigned to each phylum using the buscogenes taxrule. An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/drTriDubi3_1/dataset/drTriDubi3_1/cumulative.

Figure 5. Genome assembly of Trifolium dubium, drTriDubi3.1: Hi-C contact map of the drTriDubi3.1 assembly, visualised using HiGlass.

Chromosomes are shown in order of size from left to right and top to bottom. An interactive version of this figure may be viewed at https://genome-note-higlass.tol.sanger.ac.uk/l/?d=F0MkUMXRRMqAc9HJXoQ8LA.

Table 2. Chromosomal pseudomolecules in the genome assembly of Trifolium dubium, drTriDubi3.

INSDC
accession	Chromosome	Length
(Mb)	GC%	
OX638062.1	1	64.64	35.0	
OX638063.1	2	59.51	32.5	
OX638064.1	3	59.37	32.5	
OX638065.1	4	51.57	33.0	
OX638066.1	5	50.66	33.0	
OX638067.1	6	47.18	32.5	
OX638068.1	7	46.01	32.5	
OX638069.1	8	40.67	32.5	
OX638070.1	9	40.21	32.0	
OX638071.1	10	39.28	32.5	
OX638072.1	11	38.74	32.0	
OX638073.1	12	36.76	31.5	
OX638074.1	13	35.95	32.0	
OX638075.1	14	34.19	32.0	
OX638076.1	15	31.43	31.5	
OX638079.1	Pltd	0.13	35.0	
OX638077.1	MT1	0.13	44.5	
OX638078.1	MT2	0.18	45.5	

The estimated Quality Value (QV) of the final assembly is 67.2 with k-mer completeness of 100.0%, and the assembly has a BUSCO v5.3.2 completeness of 98.9% (single = 5.4%, duplicated = 93.6%), using the fabales_odb10 reference set ( n = 5,366).

Metadata for specimens, barcode results, spectra estimates, sequencing runs, contaminants and pre-curation assembly statistics are given at https://tolqc.cog.sanger.ac.uk/darwin/dicots/Trifolium_dubium/.

Methods

Sample acquisition, genome size estimation and nucleic acid extraction

Leaf and flower samples of Trifolium dubium were collected from Gorebridge, Scotland, UK (latitude 55.84, longitude –3.04) on 2021-08-11. One specimen was used for DNA sequencing (specimen ID EDTOL02342, ToLID drTriDubi3); another was used for Hi-C sequencing (specimen ID EDTOL02341, ToLID drTriDubi2); and a third specimen was used for RNA sequencing (specimen ID EDTOL02343, ToLID drTriDubi4). The specimens were collected and identified by Markus Ruhsam (Royal Botanic Garden Edinburgh) and preserved in liquid nitrogen. A voucher specimen from the same population of the sequenced plant is housed in the herbarium of the Royal Botanic Garden Edinburgh (E), available at https://data.rbge.org.uk/herb/E01152325.

The genome size was estimated by flow cytometry using the fluorochrome propidium iodide and following the ‘one-step’ method as outlined in Pellicer et al. (2021). 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 ‘Champion Moss Curled’ with an assumed 1C-value of 2,200 Mb ( Obermayer et al., 2002).

The workflow for high molecular weight (HMW) DNA extraction at the Wellcome Sanger Institute (WSI) includes a sequence of core procedures: sample preparation; sample homogenisation, DNA extraction, fragmentation, and clean-up. In sample preparation, the drTriDubi3 sample was weighed and dissected on dry ice ( Jay et al., 2023).

For sample homogenisation, flower and leaf tissue was cryogenically disrupted using the Covaris cryoPREP ® Automated Dry Pulverizer ( Narváez-Gómez et al., 2023). HMW DNA was extracted using the Automated Plant MagAttract v2 protocol ( Todorovic et al., 2023a). HMW DNA was sheared into an average fragment size of 12–20 kb in a Megaruptor 3 system with speed setting 30 ( Todorovic et al., 2023b). Sheared DNA was purified by solid-phase reversible immobilisation ( Strickland et al., 2023): in brief, the method employs a 1.8X ratio of AMPure PB beads to sample to eliminate shorter fragments and concentrate the DNA. The concentration of the sheared and purified DNA was assessed using a Nanodrop spectrophotometer and Qubit Fluorometer and Qubit dsDNA High Sensitivity Assay kit. Fragment size distribution was evaluated by running the sample on the FemtoPulse system.

RNA was extracted from flower tissue of drTriDubi4 in the Tree of Life Laboratory at the WSI using the RNA Extraction: Automated MagMax™ mirVana protocol ( do Amaral et al., 2023). The RNA concentration was assessed using a Nanodrop spectrophotometer and a Qubit Fluorometer using the Qubit RNA Broad-Range Assay kit. Analysis of the integrity of the RNA was done using the Agilent RNA 6000 Pico Kit and Eukaryotic Total RNA assay.

Protocols developed by the WSI Tree of Life core laboratory are publicly available on protocols.io ( Denton et al., 2023).

Sequencing

Pacific Biosciences HiFi circular consensus DNA sequencing libraries were constructed according to the manufacturers’ instructions. Poly(A) RNA-Seq libraries were constructed using the NEB Ultra II RNA Library Prep kit. DNA and RNA sequencing was performed by the Scientific Operations core at the WSI on Pacific Biosciences SEQUEL II (HiFi) and Illumina NovaSeq 6000 (RNA-Seq) instruments. Hi-C data were also generated from flower and leaf tissue of drTriDubi2 using the Arima2 kit and sequenced on the Illumina NovaSeq 6000 instrument.

Genome assembly, curation and evaluation

Assembly was carried out with HiCanu ( Nurk et al., 2020) and haplotypic duplication was identified and removed with purge_dups ( Guan et al., 2020). The assembly was then scaffolded with Hi-C data ( Rao et al., 2014) using YaHS ( Zhou et al., 2023). The assembly was checked for contamination and corrected as described previously ( Howe et al., 2021). Manual curation was performed using HiGlass ( Kerpedjiev et al., 2018) and PretextView ( Harry, 2022). The organelle genomes were assembled using MitoHiFi ( Uliano-Silva et al., 2023) and OATK ( Zhou, 2023).

A Hi-C map for the final assembly was produced using bwa-mem2 ( Vasimuddin et al., 2019) in the Cooler file format ( Abdennur & Mirny, 2020). To assess the assembly metrics, the k-mer completeness and QV consensus quality values were calculated in Merqury ( Rhie et al., 2020). This work was done using Nextflow ( Di Tommaso et al., 2017) DSL2 pipelines “sanger-tol/readmapping” ( Surana et al., 2023a) and “sanger-tol/genomenote” ( Surana et al., 2023b). The genome was analysed within the BlobToolKit environment ( Challis et al., 2020) and BUSCO scores ( Manni et al., 2021; Simão et al., 2015) were calculated.

Table 3 contains a list of relevant software tool versions and sources.

Table 3. Software tools: versions and sources.

Software tool	Version	Source	
BlobToolKit	4.1.7	https://github.com/blobtoolkit/blobtoolkit	
BUSCO	5.3.2	https://gitlab.com/ezlab/busco	
HiCanu	2.2	https://github.com/marbl/canu	
HiGlass	1.11.6	https://github.com/higlass/higlass	
Merqury	MerquryFK	https://github.com/thegenemyers/MERQURY.FK	
MitoHiFi	2	https://github.com/marcelauliano/MitoHiFi	
OATK	0.1	https://github.com/c-zhou/oatk	
PretextView	0.2	https://github.com/wtsi-hpag/PretextView	
purge_dups	1.2.3	https://github.com/dfguan/purge_dups	
sanger-tol/genomenote	v1.0	https://github.com/sanger-tol/genomenote	
sanger-tol/readmapping	1.1.0	https://github.com/sanger-tol/readmapping/tree/1.1.0	
YaHS	1.1a.2	https://github.com/c-zhou/yahs	

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 here. By agreeing with and signing up to the Sampling Code of Practice, the Darwin Tree of Life Partner agrees they will meet the legal and ethical requirements and standards set out within this document in respect of all samples acquired for, and supplied to, the Darwin Tree of Life Project.

Further, the Wellcome Sanger Institute employs a process whereby due diligence is carried out proportionate to the nature of the materials themselves, and the circumstances under which they have been/are to be collected and provided for use. The purpose of this is to address and mitigate any potential legal and/or ethical implications of receipt and use of the materials as part of the research project, and to ensure that in doing so we align with best practice wherever possible. 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.

Data availability

European Nucleotide Archive: Trifolium dubium. Accession number PRJEB59394; https://identifiers.org/ena.embl/PRJEB59394 ( Wellcome Sanger Institute, 2023). The genome sequence is released openly for reuse. The Trifolium dubium genome sequencing initiative is part of the European Reference Genome Atlas Pilot Project ( https://www.erga-biodiversity.eu/pilot-project) as well as the Darwin Tree of Life (DToL) project. 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 Table 1.

Author information

Members of the Royal Botanic Garden Edinburgh Genome Acquisition Lab are listed here: https://doi.org/10.5281/zenodo.4786682.

Members of the Plant Genome Sizing collective are listed here: https://doi.org/10.5281/zenodo.7994306.

Members of the Darwin Tree of Life Barcoding collective are listed here: https://doi.org/10.5281/zenodo.4893703.

Members of the Wellcome Sanger Institute Tree of Life Management, Samples and Laboratory team are listed here: https://doi.org/10.5281/zenodo.10066175.

Members of Wellcome Sanger Institute Scientific Operations: Sequencing Operations are listed here: https://doi.org/10.5281/zenodo.10043364.

Members of the Wellcome Sanger Institute Tree of Life Core Informatics team are listed here: https://doi.org/10.5281/zenodo.10066637.

Members of the Tree of Life Core Informatics collective are listed here: https://doi.org/10.5281/zenodo.5013541.

Members of the Darwin Tree of Life Consortium are listed here: https://doi.org/10.5281/zenodo.4783558.

10.21956/wellcomeopenres.23438.r89306
Reviewer response for version 1
Fukasawa Yoshinori 1Referee https://orcid.org/0000-0002-0882-4392

1 Center for Center for Bioscience Research and Education, Utsunomiya University, Tochigi, Japan
9 9 2024 Copyright: © 2024 Fukasawa Y
2024
https://creativecommons.org/licenses/by/4.0/ 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.
Version 1recommendationapprove
This manuscript reports the genome sequence of Trifolium dubium, the Irish Shamrock, assembled from PacBio HiFi and Hi-C data. The final assembly (drTriDubi3.1) has a total length of 679.1 Mb in 153 sequence scaffolds, with a scaffold N50 of 46.0 Mb and 99.51% of the assembled sequence assigned to 15 chromosomal-level scaffolds. The authors also report the assembly of the mitochondrial (133.86 kb and 182.32 kb) and plastid (126.22 kb) genomes.

Major Points:

- Haplotype phasing: The manuscript states that the assembly represents one haplotype and that contigs from the second haplotype were also deposited. However, the statistics for the phased assembly are not available. It would be beneficial to clarify this aspect and explain the rationale behind not aiming for a fully phased assembly.

- Chromosome 1 structure: The manuscript indicates that the order and orientation of contigs on chromosome 1 between 37.5 Mb and 42.4 Mb are uncertain. It would be beneficial to provide further detail on the difficulties encountered in this region and to present potential solutions or avenues for further investigation that could be pursued in order to resolve this ambiguity.

- Genome annotation: The manuscript makes a cursory mention of the planned use of RNA-Seq data for genome annotation and presentation through Ensemble. However, providing a more detailed rationale for the decision to extract RNA only from flower tissue would be beneficial for readers. Additionally, discussing the expected timeline for the annotation release would be helpful for researchers interested in utilizing this resource. 

Minor Points:

- It is imperative that the source of the k-mers used in Merqury be explicitly described. This issue could be addressed by specifying whether the k-mers are derived from HiFi reads. If this is the case, readers could consider that this could potentially lead to an overestimation of QV within the Merqury framework.

- Subgenome identification: The authors may employ existing genomic resources of the proposed parental species (T. campestre and T. micranthum) to identify homeologous regions within the T. dubium assembly.

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 genomics / bioinformatics

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.

10.21956/wellcomeopenres.23438.r87955
Reviewer response for version 1
Satrio Rizky Dwi 12Referee https://orcid.org/0000-0003-1716-2076

1 Department of Biology, The Republic of Indonesia Defense University, Bogor, Indonesia
2 Universitas Pertahanan Indonesia, Tajur, Indonesia
26 8 2024 Copyright: © 2024 Satrio RD
2024
https://creativecommons.org/licenses/by/4.0/ 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.
Version 1recommendationapprove
The manuscript titled "The genome sequence of lesser trefoil or Irish shamrock, Trifolium dubium Sibth. (Fabaceae)" presents a high-quality genome assembly of Trifolium dubium, a species often considered to represent the traditional Irish shamrock. The genome was sequenced as part of a collaboration between the Darwin Tree of Life and the European Reference Genome Atlas. The study reports a genome assembly size of 679.1 megabases, scaffolded into 15 chromosomal pseudomolecules, along with two mitochondrial genomes and a plastid genome.

Review

Rationale for Creating the Dataset Clarity: The manuscript clearly explains the motivation behind sequencing the Trifolium dubium genome. The species holds cultural significance and has potential agricultural importance due to its ability to fix nitrogen and produce condensed tannins, making it a valuable resource for future comparative studies and breeding programs.

Critique: The rationale is well articulated. However, the manuscript could benefit from a more detailed discussion of specific research questions that the dataset could address, particularly in comparative genomics and plant breeding, although this manuscript is a data note article.

Appropriateness of Protocols and Technical Soundness Protocols: The manuscript describes the sequencing and assembly methods in great detail. High molecular weight (HMW) DNA extraction was performed, followed by sequencing with PacBio HiFi, Hi-C, and RNA-Seq technologies. The assembly was curated using multiple tools, ensuring high quality.

Technical Soundness: The work is technically sound, with a high degree of thoroughness in the methods described. The genome assembly metrics, such as scaffold N50 and BUSCO scores, indicate a high-quality assembly.

Critique: While the protocols are appropriate and robust, it would be beneficial to include more details on the challenges faced during the assembly process and how they were addressed, especially during manual curation.

Detailing of Methods and Materials for Replication Detailing: The manuscript provides detailed descriptions of the methods, including specific protocols for DNA and RNA extraction, sequencing, and data processing. It references publicly available protocols on platforms like protocols.io, which enhances reproducibility.

Critique: The level of detail is generally sufficient for replication. However, the manuscript could improve by providing more insights into the specific conditions used during sequencing and assembly, such as environmental factors during sample collection, particularly for RNA-seq analysis.

Presentation and Accessibility of Datasets Presentation: The datasets are presented clearly, with comprehensive tables summarizing the assembly metrics, accession numbers, and scaffold information. The manuscript also provides links to interactive tools for further exploration of the data.

Accessibility: The data is openly accessible via public repositories, with clear instructions on how to access it.

Critique: The presentation is strong, but the manuscript could enhance usability by providing more user-friendly summaries or visualizations, such as a Circos diagram representing an overview figure of the genome assembly for each chromosome.

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 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.

Competing interests: No competing interests were disclosed.

Competing interests: No competing interests were disclosed.
==== Refs
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