Science, Faculty of
Science, Irving K. Barber Faculty of (Okanagan)
Non UBC
Biology, Department of (Okanagan)
Botany, Department of
DSpace
Michael Smith Laboratories
University of British Columbia. Biodiversity Research Centre
Beaty Biodiversity Museum
Hirabayashi, Kaede
Dumigan, Christopher R.
Kučka, Matúš
Percy, Diana M.
Guerriero, Gea
Cronk, Quentin C. B.
Deyholos, Michael K.
Todesco, Marco
Stinging nettles (Urtica dioica) have a long history of association with human civilization, having been used as a source of textile fibers, food and medicine. Here, we present a chromosome-level, phased genome assembly for a diploid female clone of Urtica dioica from Romania. Using a combination of PacBio HiFi, Oxford Nanopore, and Illumina sequencing, as well as Hi-C long-range interaction data (using a novel Hi-C protocol presented here), we assembled two haplotypes of 574.9 Mbp (contig N50 = 10.9 Mbp, scaffold N50 = 44.0 Mbp) and 521.2 Mbp (contig N50 = 13.5 Mbp, scaffold N50 = 48.0 Mbp), with assembly BUSCO scores of 92.6% and 92.2%. We annotated 20,333 and 20,140 genes for each haplotype, covering over 90% of the complete BUSCO genes and including two copies of a gene putatively encoding the neurotoxic peptide urthionin, which could contribute to nettle’s characteristic sting. Despite its relatively small size, the nettle genome displays very high levels of repetitiveness, with transposable elements comprising more than 60% of the genome, as well as considerable structural variation. This genome assembly represents an important resource for the nettle community and will enable the investigation of the genetic basis of the many interesting characteristics of this species.
Academic Editors: Françoise Martzand Michel ChalotReceived: 21 November 2024Revised: 24 December 2024Accepted: 2 January 2025Published: 3 January 2025Citation: Hirabayashi, K.;Dumigan, C.R.; Kucˇka, M.;Percy, D.M.; Guerriero, G.; Cronk, Q.;Deyholos, M.K.; Todesco, M.A High-Quality Phased GenomeAssembly of Stinging Nettle (Urticadioica ssp. dioica). Plants 2025, 14, 124.https://doi.org/10.3390/plants14010124Copyright: © 2025 by the authors.Licensee MDPI, Basel, Switzerland.This article is an open access articledistributed under the terms andconditions of the Creative CommonsAttribution (CC BY) license(https://creativecommons.org/licenses/by/4.0/).ArticleA High-Quality Phased Genome Assembly of Stinging Nettle(Urtica dioica ssp. dioica)Kaede Hirabayashi 1 , Christopher R. Dumigan 2 , Matúš Kucˇka 2,3 , Diana M. Percy 4,5 , Gea Guerriero 6 ,Quentin Cronk 4,5,7 , Michael K. Deyholos 2 and Marco Todesco 1,2,4,5,*1 Michael Smith Laboratories, University of British Columbia, 2185 East Mall,Vancouver, BC V6T 1Z4, Canada; kaede.hirabayashi@msl.ubc.ca2 Biology Department, University of British Columbia, 1177 Research Rd, Kelowna, BC V1V 2W9, Canada;cdumigan94@gmail.com (C.R.D.); matkucka@gmail.com (M.K.); michael.deyholos@ubc.ca (M.K.D.)3 Institute of Plant and Environmental Sciences, Faculty of Agrobiology and Food Resources,Slovak University of Agriculture in Nitra, 949 76 Nitra, Slovakia4 Botany Department, University of British Columbia, Vancouver, BC V6T 1Z4, Canada;diana.percy@botany.ubc.ca (D.M.P.); quentin.cronk@ubc.ca (Q.C.)5 Biodiversity Research Centre, University of British Columbia, Vancouver, BC V6T 1Z4, Canada6 Luxembourg Institute of Science and Technology, Technology (LIST), 5, Rue Bommel,L-4940 Hautcharage, Luxembourg; gea.guerriero@list.lu7 Beaty Biodiversity Museum, University of British Columbia, Vancouver, BC V6T 1Z4, Canada* Correspondence: mtodesco@msl.ubc.caAbstract: Stinging nettles (Urtica dioica) have a long history of association with humancivilization, having been used as a source of textile fibers, food and medicine. Here, wepresent a chromosome-level, phased genome assembly for a diploid female clone of Urticadioica from Romania. Using a combination of PacBio HiFi, Oxford Nanopore, and Illuminasequencing, as well as Hi-C long-range interaction data (using a novel Hi-C protocolpresented here), we assembled two haplotypes of 574.9 Mbp (contig N50 = 10.9 Mbp,scaffold N50 = 44.0 Mbp) and 521.2 Mbp (contig N50 = 13.5 Mbp, scaffold N50 = 48.0 Mbp),with assembly BUSCO scores of 92.6% and 92.2%. We annotated 20,333 and 20,140 genes foreach haplotype, covering over 90% of the complete BUSCO genes and including two copiesof a gene putatively encoding the neurotoxic peptide urthionin, which could contributeto nettle’s characteristic sting. Despite its relatively small size, the nettle genome displaysvery high levels of repetitiveness, with transposable elements comprising more than 60% ofthe genome, as well as considerable structural variation. This genome assembly representsan important resource for the nettle community and will enable the investigation of thegenetic basis of the many interesting characteristics of this species.Keywords: genome assembly; Urtica dioica; stinging nettle; trichomes; fiber crops; chromosomalstructural variation; Hi-C protocol1. IntroductionUrtica dioica L. (U. dioica ssp. dioica; stinging nettle, or common nettle) is an herbaceousperennial in the Urticaceae family, native to Eurasia and northwest Africa [1]. U. dioicais widely distributed in temperate and tropical climates and can grow on a variety ofdifferent soil types, although it prefers moist habitats rich in nitrogen and phosphorus [2].It is often found on disturbed soils, where it can grow in dense stands, which has earnedU. dioica a reputation as a weed. However, since at least the Bronze Age and continuingto the present day, U. dioica and its close relatives have also been used by humans assources of fiber, food, and medicine [3–5]. Similar to cannabis (Cannabis sativa L.), flaxPlants 2025, 14, 124 https://doi.org/10.3390/plants14010124Plants 2025, 14, 124 2 of 18(Linum usitatissimum L.), and ramie (Boehmeria nivea (L.) Gaudich.), the stems of U. dioicaproduce long (43–58 mm) bast fibers that are rich in crystalline cellulose (>70%) and have arelatively low lignin content (2–7%) [6]. Fibers have been used historically in papermaking,cordage, and textiles, and there is renewed interest in using nettle in both clothing andcomposite materials. Both the shoots and roots of stinging nettle are consumed in somecultures, and dried nettle powder contains an impressive 30% protein content (w/w) [7].The lipids produced in the leaves also contain a very high proportion of polyunsaturatedfatty acids (41% alpha-linolenic acid, 18:3n − 3; 12% linoleic acid, 18:2n − 6), which arebeneficial to human nutrition [8]. Teas and other extracts made from stinging nettle havebeen reported to have anti-proliferative, antibacterial, anti-inflammatory, hypoglycemic,and other pharmacological activities [9].The common name of stinging nettle comes from the irritation experienced by verte-brates when they make skin contact with certain trichomes of U. dioica. Although this effectwas previously attributed to the presence of histamines, organic acids, and neurotransmit-ters [10], it has recently been demonstrated that small peptides, namely urthionin (∆-Uf1a)and a sodium-gated channel modulator named urticatoxin (β/δ-Uf2a), contribute to thepainful sensation [11]. Small neurotoxic peptides in Dendrocnide species similarly affectsodium-gated channels [12].U. dioica is either diploid (2n = 24, 26) or tetraploid (2n = 48, 52), with tetraploids beingmuch more abundant [2,13]. Occasionally, triploid and pentaploid individuals are foundwithin populations [2]. Genome size estimates for diploid U. dioica also vary accordingto different studies, ranging from 558 Mbp to 660 Mbp [2,13]. U. dioica is distinct fromslender nettle, Urtica gracilis Aiton (also known as U. dioica ssp. gracilis (Aiton) Selander),which is native to North America. While U. dioica is generally dioecious, meaning thatfemale and male flowers are on separate individuals, U. gracilis is monoecious, with femaleand male flowers on the same plant [14]. Complex variation of morphological traits inthe genus Urtica makes the classification of species and subspecies sometimes difficult,although attempts have been made to resolve these phylogenetic relationships using molec-ular data [15]. More recently, reference genome assemblies have been produced for a fewspecies in the Urticaceae family; these include U. urens L. [16] and Parietaria judaica L. [17],as well as an assembly produced by the Darwin Tree of Life project [18] for an U. dioica L.individual collected in the UK (Table S1). While these assemblies provide very valuableresources for the research community, they are not fully haplotype-resolved, meaning thatthey report only one chromosome-level primary haplotype, with contigs belonging to theother haplotype(s) being collected in a shorter secondary haplotype assembly. A high-quality haplotype-resolved reference genome of a well-characterized U. dioica individualwould allow for accurate comparison of sequence and structural diversity within haplo-types, further facilitating the study of the phenotypic, genetic, and karyotypic diversitywithin U. dioica and helping improve our understanding of taxonomic relationships withinthe Urticaceae.In this study, we develop a diploid, phased chromosome-level genome assembly forUrtica dioica ssp. dioica using a combination of sequencing approaches, including PacBioHiFi and Oxford Nanopore (ONT) long reads, whole genome shotgun (WGS) Illumina shortreads, and chromatin conformation capture (Hi-C). We further explore characteristics ofthe stinging nettle genome, such as the repeat landscape across chromosomes, the presenceof large structural variants (SVs) between haplotypes, and how it compares to related taxa.Finally, we comment on the presence of putative neurotoxic peptides associated with thenettle’s sting.Plants 2025, 14, 124 3 of 182. Results and Discussion2.1. Genome Sequencing and AssemblyWe generated 66.3 Gbp of PacBio HiFi data (115X genome coverage) from a female,diploid individual of stinging nettle. HiFi reads had a median read length of 15.45 kbpand a median read quality of Q29. Only reads with quality ≥ Q20 were used for theassembly (50.7 Gbp, 88X genomic coverage). We also generated 29.02 Gbp paired-endHi-C reads (50X genome coverage), with 96.49% of the reads with quality > Q30. Thecombination of long reads and long-range interaction data produced an initial phasedcontig-level assembly. Haplotype 1 (H1) had a total length of 574.795 Mbp and contigN50 = 20.587 Mbp (1404 contigs); haplotype 2 (H2) had a total length of 521,297 Mbp andN50 = 24.787 Mbp (229 contigs). There is a considerable range of genome size estimates forUrtica dioica from previous studies [3,13], so we performed confirmatory flow cytometryon the individual that we sequenced, which was originally estimated at 1C = 650 Mb [13].Our new estimate, reported here, is 2C = 1.26 pg, which translates to 1C = 616 Mbp. Whileour assembled genome is smaller than that estimate, it has high BUSCO completenessscores (>92% for each haplotype) and kmer completeness (98.15% for both haplotypescombined; Table S2, Figure S1), consistent with recent suggestions that flow cytometryover-estimates genome sizes [19]. Scaffolding using Hi-C data anchored contigs into13 pseudochromosomes for each haplotype. We visually inspected Hi-C contact maps forthe two haplotypes and manually corrected misjoins and misassemblies, retaining onlyputative SVs whose presence was strongly supported by Hi-C data and by the presence oflong HiFi and ONT reads spanning their borders. Finally, we compared the chromosomeorganization of the haplotype assemblies to that of the U. dioica assembly produced bythe Darwin Tree of Life project [1] to ensure that Hi-C contact maps supported our contigordering (Figure S2).The final H1 assembly had a total length of 574.93 Mbp, a scaffold N50 = 43.96 Mbp,and a contig N50 = 10.89 Mbp, where 92.59% of the contigs were above 50 kbp. Thefinal H2 assembly was of similar quality, with a total length of 521.16 Mbp, a scaffoldN50 = 47.99 Mbp, and a contig N50 = 13.53 Mbp, with 98.98% of contigs above 50 kbp inlength. The BUSCO score was 92.6% complete for H1 and 92.2% complete for H2, with verylow levels of duplication (<3%) (Table 1; Figure 1). While 89.5% and 97.8% of the H1 andH2 genome were placed in the chromosomes, respectively, we had 72.33 Mbp of unplacedsequences. We note that 95.44% of these sequences (69.0 Mbp) were repetitive, and weonly detected five BUSCO genes within them. Hifiasm placed all the repetitive sequencesthat could not be unequivocally assigned to either haplotype in H1, which explains thedifference in size between H1 and H2.2.2. Genome StructureWe expected to find very high synteny between the two haplotypes of our stingingnettle assembly, especially since we had high HiFi long-reads and Hi-C coverage, and thor-ough manual curation should minimize the chances of misassemblies between haplotypes.However, we observed numerous SVs between haplotype assemblies (see chromosomes 1,2, 3, and 8 in Figures 2 and S3). In particular, we found that the two haplotypes differed bya massive inversion (18.4 Mbp in length) on chromosome 8, which was surrounded by mul-tiple duplicated regions (Figure 3). We manually checked all those SVs using Hi-C data andlooked for HiFi and Nanopore reads spanning their breakpoints; this included manually“correcting” these SVs in either haplotype to determine if this resulted in improved Hi-Ccontact maps. In all cases, these checks supported the current chromosome organization ofthe two haplotypes (Figure 2, Tables S3 and S4).Plants 2025, 14, 124 4 of 18Table 1. Genome assembly statistics. Heterozygosity values were obtained from short-read whole-genome sequencing data generated ad hoc.Haplotype 1 Haplotype 2Parameters Contig Scaffold Contig ScaffoldTotal length (bp) 574,934,600 521,157,583Contig/Scaffold number 1459 1598 376 248N50 (Mbp) 10.89 43.96 13.53 47.99% main genome in scaffolds > 50 kbp 92.59 98.98% of genome anchored to 13 chromosomes 89.52 97.83BUSCO (C%) 92.6 92.2BUSCO (S%) 90.5 90.1BUSCO (D%) 2.1 2.1QV score (Merqury) 42.05 44.45kmer completeness (%; Merqury) 81.72 81.61Number of protein-coding genes annotated 20,333 20,140Protein BUSCO (Complete %) 90.5 90.4TE coverage (%) 69.1 68.6Whole genome LAI 16.96 11.15Heterozygosity (%) 1.53Plants 2025, 14, x FOR PEER REVIEW 4 of 18 kmer completeness (%; Merqury) 81.72 81.61 Number of protein-coding genes annotated 20,333 20,140 Protein BUSCO (Complete %) 90.5 90.4 TE coverage (%) 69.1 68.6 Whole genome LAI 16.96 1 .15 Heterozygosity (%) 1.53 Figure 1. Haplotype-resolved assembly of a female Urtica dioica ssp. dioica individual (2n = 26). The two haplotypes are compared (H1 on the right, blue; H2 on the left, yellow). The tracks in the Circos plot represent (a) aligned regions between haplotypes, (b) gene density, (c) TE density, (d) repeats, Shannon diversity score, and (e) predicted centromeric regions, highlighted in black on the ideo-gram. 2.2. Genome Structure We expected to find very high synteny between the two haplotypes of our stinging nettle assembly, especially since we had high HiFi long-reads and Hi-C coverage, and thorough manual curation should minimize the chances of misassemblies between hap-lotypes. However, we observed numerous SVs between haplotype assemblies (see chro-mosomes 1, 2, 3, and 8 in Figures 2 and S3). In particular, we found that the two haplotypes differed by a massive inversion (18.4 Mbp in length) on chromosome 8, which was sur-rounded by multiple duplicated regions (Figure 3). We manually checked all those SVs using Hi-C data and looked for HiFi and Nanopore reads spanning their breakpoints; this included manually “correcting” these SVs in either haplotype to determine if this resulted in improved Hi-C contact maps. In all cases, these checks supported the current chromo-some organization of the two haplotypes (Figure 2, Tables S3 and S4). Figure 1. Haplotype-resolved assembly of a female Urtica dioica ssp. dioica individual (2n = 26). Thetwo haplotypes are compared (H1 on the right, blue; H2 on the left, yellow). The tracks in the Circosplot represent (a) aligned regions between haplotypes, (b) gene density, (c) TE density, (d) repeats,Shannon diversity score, and (e) predicted centromeric regions, highlighted in black on the ideogram.Plants 2025, 14, 124 5 of 18Plants 2025, 14, x FOR PEER REVIEW 5 of 18 Figure 2. Chromosome structure of the U. dioica genome (a) Hi-C contact map of haplotype H1 and (b) alignment between haplotype H1 and H2 of the U. dioica ssp. dioica genome assembly presented in this study. Only the 13 chromosomes are shown in (a); additional smaller contigs were not plotted for clarity. In (b), blue represents forward strand alignment, and red represents reverse strand align-ment. Figure 3. The 18.4 Mbp inversion found between haplotypes on chromosome 8 is supported by Hi-C data. In the left panel, Hi-C data are aligned to both haplotypes simultaneously to create a haplo-type-aware heatmap. Green lines represent contigs. Only the section of the genome-wide haplotype-aware heatmap corresponding to chromosome 8 is shown; top left contigs correspond to the H1 and bottom right contigs correspond to the H2 versions of chromosome 8. The right panel shows changes in the contact map when Hi-C reads are mapped to modified versions of the assembly, in which the orientation of the large putative inversion on chromosome 8 is flipped in H1 (top) or H2 (bottom). Disruption of (haplotype-aware) Hi-C patterns in both of these cases supports the presence of op-posite orientations of the inversion in the two haplotypes. We also observed fragmented alignments between haplotypes in regions with low gene density and high repetitiveness in several chromosomes (compare panels a–d in Fig-ure 1). Across all 13 chromosomes, SyRI classified only about 71% of the regions between the two haplotypes as syntenic, with around 16% of the genome remaining unaligned Figure 2. Chromosome structure of the U. dioica genome (a) Hi-C contact map of haplotype H1 and(b) alignment between haplotype H1 and H2 of the U. dioica ssp. dioica genome assembly presented inthis study. Only the 13 chromosomes are shown in (a); additional smaller contigs were not plotted forclarity. In (b), blue represents forward strand alignment, and red represents reverse strand alignment.Plants 2025, 14, x FOR PEER REVIEW 5 of 18 Figure 2. Chromosome structure of the U. dioica genome (a) Hi-C contact map of haplotype H1 and (b) alignment between haplotype H1 and H2 of the U. dioica ssp. dioica genome assembly presented in this study. Only the 13 chromosomes are shown in (a); additional smaller contigs were not plotted fo clarity. In (b), blu represents forward strand alig ment, nd red repr sents r verse strand align-ment. Figure 3. The 18.4 Mbp inversion found between haplotypes on chromosome 8 is supported by Hi-C data. In the left panel, Hi-C data are aligned to both haplotypes simultaneously to create a haplo-type-aware heatmap. Green lines represent contigs. Only the section of the genome-wide haplotype-aware heatmap corresponding to chromosome 8 is shown; top left contigs correspond to the H1 and bottom right contigs correspond to the H2 versions of chromosome 8. The right panel shows changes in the contact map whe Hi-C reads are mapp d to modified versions f the ass mbly, in which the orientation of the large putative inversion on chromosome 8 is flipped in H1 (t p) or H2 (bottom). Disruption of (h lotype-aware) Hi-C patterns in both of t se cases supports the presence of op-posite orientations of the inversion in the two haplotypes. We also observed fragmented alignments between haplotypes in regions with low gene density and high repetitiveness in several chromosomes (compare panels a–d in Fig-ure 1). Across all 13 chromosomes, SyRI classified only about 71% of the regions between the two haplotypes as syntenic, with around 16% of the genome remaining unaligned Figure 3. The 18.4 Mbp inversion found between haplotypes on chromosome 8 is supported byHi-C data. In the left panel, Hi-C data are aligned to both ha lotypes simultaneously to create ahaplotype-aware heatmap. Green lines represent contigs. Only the section of the genome-widehaplotype-aware heatmap corresponding to chromosome 8 is shown; top left contigs correspond tothe H1 and bottom right contigs correspond to the H2 versions of chromosome 8. The right panelshows changes in the contact map when Hi-C reads are mapped to modified versions of the assembly,in which the orientation of the large putative inversion n chromosome 8 is flipped in H1 (top) or H2(bottom). Disruption of (haplotype-aware) Hi-C patterns in both of these cases supports the presenceof opposite orientations of the inversion in the two haplotypes.Plants 2025, 14, 124 6 of 18We also observed fragmented alignments between haplotypes in regions with low genedensity and high repetitiveness in several chromosomes (compare panels a–d in Figure 1).Across all 13 chromosomes, SyRI classified only about 71% of the regions between thetwo haplotypes as syntenic, with around 16% of the genome remaining unaligned (Table S5)due to their extremely repetitive content (see below). While it is not possible to completelyexclude that some of these patterns are due to artifacts in sequencing or assembly, theseresults highlight a high occurrence of structural variation in the stinging nettle genome(Figures 1a, 2b and S2). Structural variation between haplotypes, especially if they havebeen maintained in nettle for many generations, could also contribute to the high levelof estimated heterozygosity (~1.5%), which was obtained from short read sequence datafrom the same stinging nettle individual and was therefore determined independentlyfrom the genome assembly. While the report of such extensive structural variation in thegenome of nettle is novel, the importance of SVs in maintaining sequence and functionaldiversity in wild [20] and cultivated species [21] is being increasingly recognized. Giventhe wide range of ecosystems in which stinging nettles grow [2], it is tempting to speculatethat these SVs could be involved in adaptation to particular environments, as is the casein other systems [22,23]. However, additional experimental evidence and species-widegenetic and phenotypic analyses will be required to assess the adaptive relevance of SVsin nettle.2.3. Genes and Repeats LandscapeGene annotation with the BRAKER3 pipeline identified 20,333 and 20,140 genes onH1 and H2, respectively, with annotation BUSCO scores of 90.5% for H1 and 90.4% for H2,showing high levels of completeness (Figure 1b, Tables 1 and S6). Ab initio and homology-based gene predictions were complemented with RNAseq data for three different tissuesof nettle, obtained from a previous study [24]; while this RNAseq dataset is unlikely toinclude the entirety of transcribed genes in the nettle genome, it provides additional ex-perimental support for our gene annotation. Across the entire genome, including contigsthat could not be placed on the 13 nettle chromosomes, transposable elements (TEs) ac-counted for 69.14% and 68.59% of the total sequence of H1 and H2, respectively (Table 1).Within the 13 chromosomes, we found that the most abundant repeats were Long Termi-nal Repeat retrotransposons (LTRs), which collectively covered 47.23% and 50.51% of thetwo assemblies, and Terminal Inverted Repeats (TIRs), which accounted for 15.63% and13.24% of the chromosomes (Table S7). The LTR Assembly Index (LAI) values were16.96 and 11.15 for H1 and H2, respectively, which are comparable to previously bench-marked high-quality genome assemblies [25]. TE density in 500 kbp windows varied from0 to >6000 TEs per window; however, most windows contained <1000 TEs, except for37 windows containing more than 2000 TEs (Figure 1c, Table S7).Scans for patterns of tandem repeats across the genome using RepeatOBserver [26] alsoidentified these TE clusters. RepeatOBserver also calculates a repeats Shannon diversityindex, which describes the diversity of tandem repeats across the genome, and uses itto identify the putative location of centromeres in chromosomes. Across most species,centromeric regions have been shown to have high repeat levels but low repeat diversity,and to correspond to minima for the repeats Shannon diversity index (meaning that most ofthe sequence in these regions is made up of only one or few very abundant repeats). Theseresults can also be confirmed through visual inspection of Fourier transform repeat heatmap,in which discrete banding identifies long stretch of tandem repeats typically associated withcentromeres (see, for example, heatmaps for metacentric Arabidopsis thaliana (L.) Heynh.and holocentric Morus notabilis C.K. Schneid. chromosomes, Figure 4a,b [26–28]). In ourU. dioica assemblies, repeat patterns are consistent with the presence of acrocentric orPlants 2025, 14, 124 7 of 18near telocentric centromeres in five out of 13 chromosomes (chromosomes 8, 9, 11, 12,13). For eight more chromosomes (chromosomes 1–7, 10) we observed more diffused andfragmented patterns of repeats over a large proportion of the chromosome, suggesting thepresence of polycentric centromere (Figures 1a, 4c and S4).Plants 2025, 14, x FOR PEER REVIEW 7 of 18 Figure 4. Fourier transform spectra of repeats occurrence in (a) Arabidopsis thaliana chromosome 5 with metacentric centromeric signal; (b) Morus notabilis chromosome 2, representative of a holocen-tric chromosome (Elphinstone et al., 2023 [26]); and (c) U. dioica ssp. dioica chromosome 1 (this study). Colour intensity corresponds to the number of times a specific repeat is found in a 5 kbp window. Bright horizontal lines indicate presence of a repeat sequence that repeats itself many times across that region of the chromosome, such as tandem repeats found in telomeric and centromeric regions. Multiple bands in those regions represent harmonics of the base repeat sequence. While acrocentric centromeres have been previously observed in the genome of an-other member of the Urticaceae (ramie, Boehmeria nivea (L.) Gaudich.; [29]) the presence of polycentric centromeres has not been reported in this family; however, polycentric cen-tromeres have been described in the Moraceae, which are the closest family to the Urtica-ceae [28]. The position of the predicted centromeres in the U. dioica genome is also con-sistent with those regions having the lowest gene density in the chromosome, as it is typ-ical of centromeres (Figure 1b,e; Table S8). However, it should be noted that these analyses only provide a putative centromere location, and that definitive identification of centro-mere regions would require direct experimental evidence (e.g., localization of centromeric histones H3, CENH3). 2.4. Genome Evolution The haploid chromosome number of U. dioica has been variously reported to be 12 or 13 chromosomes [30]. In this study, we unequivocally identify 13 separate chromosomes Figure 4. Fourier transform spectra of repeats occurrence in (a) Arabidopsis thaliana chromosome5 with metacentric centromeric signal; (b) Morus notabilis chromosome 2, representative of a holocen-tric chromosome (Elphinstone et al., 2023 [26]); and (c) U. dioica ssp. dioica chromosome 1 (this study).Colour intensity corresponds to the number of times a specific repeat is found in a 5 kbp window.Bright horizontal lines indicate presence of a repeat sequence that repeats itself many times acrossthat region of the chromosome, such as tandem repeats found in telomeric and centr meric regions.Multiple bands in those regions represent harmonics of the base repeat sequence.Plants 2025, 14, 124 8 of 18While acrocentric centromeres have been previously observed in the genome of an-other member of the Urticaceae (ramie, Boehmeria nivea (L.) Gaudich.; [29]) the presenceof polycentric centromeres has not been reported in this family; however, polycentriccentromeres have been described in the Moraceae, which are the closest family to theUrticaceae [28]. The position of the predicted centromeres in the U. dioica genome is alsoconsistent with those regions having the lowest gene density in the chromosome, as itis typical of centromeres (Figure 1b,e; Table S8). However, it should be noted that theseanalyses only provide a putative centromere location, and that definitive identificationof centromere regions would require direct experimental evidence (e.g., localization ofcentromeric histones H3, CENH3).2.4. Genome EvolutionThe haploid chromosome number of U. dioica has been variously reported to be 12 or13 chromosomes [30]. In this study, we unequivocally identify 13 separate chromosomesin the U. dioica genome assembly. However, base chromosome number is highly variablewithin the Urticaceae (ranging from 7 to 14 [31]), as well as within Urtica species [32]. Whilethe Urticaceae family has a complex phylogeny, multiple sources support a monophyleticorigin and subdivision in four clades (I-IV [33,34]). The divergence time between cladesII/III and clades I/IV was estimated to be 84.87 million years, and the Urticaceae familysplit from the nearest relative Moraceae 100.01 million years ago [33]. To understand theevolution of genome organization in this family, we compared our U. dioica (clade III)genome assembly to those of three other species in the Urticaceae: Urtica urens (cladeIII), Boehmeria nivea (clade I), and Parietaria judaica (clade I). Despite evidence of abundantlarge-scale chromosome rearrangements, we found synteny to be quite conserved acrossall of these species (Figure 5).Plants 2025, 14, x FOR PEER REVIEW 8 of 18 in the U. dioica genome as embly. However, base chromosome number is highly variable within the (r ging from 7 to 14 [31]), as well as within Urtica species [32]. W ile the Urticaceae family has a complex phylogeny, multiple sources support a mono-phyletic origin and subdivision in four clades (I-IV [33,34]). The divergence time between clades II/III an clades I/IV was estimated to be 84.87 million years, and the Urticaceae family split from th nearest relative Moraceae 100.01 million years ago [33]. To u er-stand the evolution of genome organizat on in this family, w compare ur U. ioica (clade III) genome assembly t those of three oth r species in the Urticaceae: Urtica urens (clade III), Boehmeria nivea (cl de I), and Pariet ri judaica (clade I). Despite evidence of abundant large-scale chromosome rearrangements, we found synteny to be quite con-served acros all of these species (Figure 5). Figure 5. Comparison of chromosome organization in four species of the Urticaceae family. Esti-mated divergence times are based on [33]. Note that while the Boehmeria nivea genome is supposed to have 14 chromosomes, the 15th largest scaffold in the assembly contained more than 300 genes and was therefore included in the figure. While the placement of that section of the genome is quite variable across species, comparison with Urtica spp. suggests that it might be part of chromosome 12 in B. nivea. Interestingly, while the phylogenetic distance between B. nivea or P. judaica and Ur-tica species is comparable, chromosomal synteny is much higher between Urtica and B. nivea than between either of them and P. judaica. Base chromosome number in U. urens and many other related Urtica species is often n = 12 [31,32], whereas U. dioica is almost always n = 13 in diploid cytotypes, while n = 12 is more frequently observed in tetraploids [13]. We find that the U. dioica chromosomes 8 and 13 are syntenic with chromosome 2 in U. urens, highlighting a clear history of chromosome fusion/fission (Figure 5). The insta-bility of the organization of these chromosomes within U. dioica could explain the high level of structural variation that we observe between haplotypes in chromosome 8 of our assembly (Figure 3, Figure S3). As more genomes in this family become available, it will be interesting to further investigate the reasons for this high flexibility in chromosome arrangement between and within related species in the Urticaceae. Figure 5. Comparison of chromosome organization in four species of the Urticaceae family. Estimateddivergence times are based on [33]. Note that while the Boehmeria nivea genome is supposed tohave 14 chromosomes, the 15th largest scaffold in the assembly contained more than 300 genes andwas therefore included in the figure. While the placement of that section of the genome is quitevariable across species, comparison with Urtica spp. suggests that it might be part of chromosome12 in B. nivea.Plants 2025, 14, 124 9 of 18Interestingly, while the phylogenetic distance between B. nivea or P. judaica and Urticaspecies is comparable, chromosomal synteny is much higher between Urtica and B. niveathan between either of them and P. judaica. Base chromosome number in U. urens and manyother related Urtica species is often n = 12 [31,32], whereas U. dioica is almost always n = 13 indiploid cytotypes, while n = 12 is more frequently observed in tetraploids [13]. We find thatthe U. dioica chromosomes 8 and 13 are syntenic with chromosome 2 in U. urens, highlightinga clear history of chromosome fusion/fission (Figure 5). The instability of the organization ofthese chromosomes within U. dioica could explain the high level of structural variation thatwe observe between haplotypes in chromosome 8 of our assembly (Figures 3 and S3). Asmore genomes in this family become available, it will be interesting to further investigatethe reasons for this high flexibility in chromosome arrangement between and within relatedspecies in the Urticaceae.2.5. Putative Neurotoxic Nettle Sting PeptidesNettle owes its common name to its sting, which constitutes an effective defencemechanism against herbivores. This is due to the presence, on the leaves and stems, of brittleneedle-like trichomes, which break upon contact and release pain-inducing chemicals [35].While the physiological mechanism of the nettle’s sting has been extensively studied sincethe early 1940s, the pain-inducing compounds were only recently identified. Initial study byFu et al. [10] showed that simple acids, including oxalic acid, tartaric acid, and formic acid,which are potentially irritant to animals, are the dominant compounds in stinging hairs,suggesting that they could be the pain-inducing compounds in nettle, and rejecting earlierhypotheses [35–37]. More recently, small neurotoxic peptides were shown to play a majorrole in causing stinging pain. In particular, two classes of such peptides were described inUrtica spp. [11]. One is a 42 amino acid-long peptide (4.3-kDa) named urthionin, whichhas cytolytic activity and a structure that resembles a known group of plant toxins calledthionins, known to disrupt the cell membrane [38]. The other is a 63 amino acids-longpeptide (6.7-kDa) called urticatoxin, which has neurotoxic activity and is seemingly specificto species in the Urticaceae tribe, such as species in the Urtica and Dendrocnide genera.Urticatoxin was originally described in U. ferox (stinging tree) and was found to inducemore severe pain than urthionin. The neurotoxicity of urticatoxin was shown to be dueto its ability to modulate the activity of vertebrate ion-gated sodium channels, similar tothe gympietides (i.e., Excelsatoxin and Moroidotoxin) previously described in Dendrocnidespecies [12]. Despite their similar effect, urticatoxins and gympietides appear to haveevolved independently, given their structural differences [11].Using a homology-based approach, we identified two copies of a gene putativelyencoding the urthionin peptide on chromosome 9 (86% amino acid match to the U. feroxmature peptide: ∆-Uf1a). The two paralogs were positioned right next to each other withthe same sequences, indicating a recent gene duplication event. Hits for urticatoxin onchromosomes 6 and 9 were only a 26–49% match to the peptides identified in U. ferox andin Dendrocnide species (Table 2), suggesting that this class of peptides might not be found inU. dioica. No genes with similarity to two other neurotoxic peptides identified in Dendrocnidespecies (Excelsatoxin A and Moroidotoxin A) were identified in the nettle genome.Plants 2025, 14, 124 10 of 18Table 2. Identification of U. dioica homologs of pain-inducing peptides identified in other stingingspecies. Nt. = nucleotides; Aa = amino acids.Peptide Name Species Described Nt. Length Aa Length Source U. dioica Genome Position Nt. Matches Aa Matches Annotated mRNA IDChr. Start EndUrthionin A(∆-Uf1a) Urtica ferox 126 42 [11] 09_H1 19832138 19832264 108 36 NA09_H1 19823191 19823317 108 36 NA09_H2 19679225 19679351 108 36 NA09_H2 19670282 19670408 108 36 g13644.t1Urticatoxin(β/δ-Uf2a) Urtica ferox 189 63 [11] 06_H1 4057458 4057644 90 30 NA06_H2 4669732 4669918 90 30 NAUrticatoxin(β/δ-Uf2b) Urtica ferox 189 63 [11] 06_H1 4057458 4057644 93 31 NA06_H2 4669732 4669918 93 31 NAUrticatoxin(β/δ-De2a) Dendrocnide excelsa 180 60 [11] NAUrticatoxin(β/δ-Dm2a) Dendrocnide moroides 183 61 [11] 09_H1 27999734 27999800 48 16 g14471.t109_H2 27490551 27499780 78 26 NAExcelsatoxin A Dendrocnide excelsa 105 35 [12] NAMoroidotoxin A Dendrocnide moroides 105 35 [12] NA3. Materials and Methods3.1. Plant Materials and SequencingWe collected a diploid female individual of Urtica dioica ssp. dioica from beside theRiver Jiu, north of Rovinari, Romania, and cultivated it in Vancouver, British Columbia (asclone 11–4). Young leaves were collected and flash-frozen in liquid nitrogen for extractions.A voucher specimen was deposited in the herbarium of the Beaty Biodiversity Museum atthe University of British Columbia (UBC).Estimation of nuclear DNA content was achieved using an Attune NxT Flow Cy-tometer (Thermo Fisher Scientific) at the University of British Columbia; preparation ofsamples followed the protocol in [39] using a Tris-MgCl2 lysis buffer [40]. RNA wasremoved with RNase A prior to staining with propidium iodide [39], and a tomato(Solanum lycopersicum L.) sample prepared with the same method was used as a standardwhen estimating the nettle genome size.High-molecular-weight DNA was extracted using a modified CTAB method [41]. APacBio HiFi sequencing library was prepared and sequenced on a Revio instrument byNovogene (San Diego, CA, USA). HiFi reads were quality-controlled using SMRT toolsv13.0.0, runqc-reports (PacBio, 2024). Whole genome shotgun sequencing (WGS) wasperformed on an Illumina HiSeq 2000 platform by CD Genomics (Shirley, NY, USA) toproduce 150 bp paired-end reads. DNA for Nanopore sequence was extracted using aThermoFisher MagMax Plant DNA kit, (Waltham, MA, USA) and then further purifiedusing a Qiagen DNeasy PowerClean column (Hilden, Germany). Nanopore sequencinglibraries were generated from HMW DNA using the Genomic DNA by Ligation (SQK-LSK110) protocol. Sequencing was carried out with FLO-FLG001 R9.4.1 flow cells on aMinION instrument. The resulting fast5 files were subsequently basecalled using Guppy6.0.1 Superior Basecalling Algorithm (dna_r9.4.1_450bps_sup.cfg) conducted on an NVIDIA3060ti. Reads with a Q score below 9 were discarded.A Hi-C library was prepared with modifications. In brief, ground frozen tissue wascross-linked in a 1.5% formaldehyde solution containing protease inhibitor. Followingnuclei isolation, chromatin was fragmented through DpnII digestion. After end-filling inthe presence of biotinylated dATP, blunt ends were ligated, and DNA was extracted. Threeand a half µg of DNA were sheared using ultrasonication (Covaris, Woburn, MA, USA),Plants 2025, 14, 124 11 of 18and fragments in the 300–500 bp size range were further selected using SPRI beads [42].Biotinylated fragments were pulled down using streptavidin-coated beads (Invitrogen,Waltham, MA, USA), and Illumina libraries were prepared following Todesco et al. [43].The Hi-C method used here combined elements from various previously published Hi-C protocols (including [44,45]) and has been optimized to work on a variety of plantspecies and to reduce library preparation costs. A detailed protocol is provided in theSupplementary Material. The resulting libraries were sequenced by Novogene (San Diego,CA, USA) on an Illumina NovaSeq X Plus instrument (Illumina Inc., San Diego, CA, USA)to generate 150 bp paired-end reads.3.2. De Novo Assembly and Quality EvaluationWe tested multiple assemblers, assembly parameters and versions to determinewhat worked best for our nettle assembly, and selected the approach that produced themost complete kmer representations in both haplotypes, based on a kmer plot analysiswith Merqury [46]. PacBio HiFi reads were first filtered by mean Q score > 20 usingfastq.filter -e 0.01 (https://github.com/LUMC/fastq-filter (accessed 15 March 2024)). Aninitial contig-level genome assembly was produced with hifiasm v0.19.8-r-603, integratingHi-C reads using the --h1 and --h2 options [47] and keeping default values for the remainingparameters. This resulted in two separate haplotype assemblies. Juicer v1.9.9 [48] and3D-DNA v180419 [49] were used to map Hi-C reads and create a contact map, which wasused to manually sort and orient the contigs to produce a chromosome-scale assemblyfor each haplotype in Juicebox v 1.11.08 [50]. To verify the positions and orientation ofcontigs, we aligned the two haplotypes and compared syntenic regions. Alignments wereperformed using minimap2 v2.28 [51,52] and regions harboring putative structural variants(SVs) were surveyed for obvious misassemblies on the Hi-C contact map. For regionsshowing ambiguous patterns in the Hi-C contact alone, we used Synteny RearrangementIdentifier (SyRI) v1.7.0 [53] to obtain precise coordinates of the putative SVs. SVs largerthan 10,000 bp were then manually reoriented in individual haplotypes to determine ifthat would improve the Hi-C contact profiles in the region (Table S3). SVs smaller than10,000 bp could not be visually assessed on Juicebox and were therefore omitted from thismanual curation step. To check whether these putative SVs were supported by the presenceof long reads spanning the expected breakpoints (following [23]), we then mapped filteredHiFi reads and ONT reads ≥30 kbp to the assembled genome using winnowmap v2.03 [54]and visualized the resulting alignments on IGV (Table S4). We used this evidence to curateboth haplotype assemblies and correct likely misassemblies. Furthermore, we re-mappedthe Hi-C reads to produce a haplotype-aware H1 + H2 contact map using 3D-DNA withparameter -q 0 [49] to finalize the assembly, which allowed for visualization of reads thatwere mapped to multiple regions in the genome (i.e., mapping quality of 0). This allowed toresolve highly repetitive regions that previously showed no visible interactions in Juicebox.However, we note that this final step fragmented our genomes into smaller contigs as atrade-off for smoother Hi-C contact map patterns. We then aligned our assemblies to theprimary haplotype of a published U. dioica assembly (NCBI accession: GCA_964188135.1,hereafter presented as Udio_DToL) to check for synteny, and we assigned correspondingchromosome numbers. To verify whether differences in chromosome organization withrespect to Udio_DToL could reflect misassemblies in our haplotypes, we re-ordered ourcontigs based on Udio_DToL using Ragtag v2.1.0 [55] (command: ragtag.py correct + rag-tag.py scaffold), re-mapped our Hi-C reads and generated a new contact map on Juicebox.If our assembly is a better representation of the real order and orientation of the contigs forour sequenced individual, regions that have a different organization in this reference-basedscaffolding assembly should appear as misassemblies in the Hi-C contact map (Figure S3).Plants 2025, 14, 124 12 of 18Genome assembly statistics were assessed with Bandage v0.8.1 [56], BBmap v39.06 [57],and BUSCO v5.1.2 (dataset: eudicot_odb10 [58,59]). A significant portion of contigs couldnot be placed on the chromosomes; while they lacked Hi-C interactions with chromosomes,they showed strong interactions with themselves on Hi-C contact maps. To assess whetherthese unplaced contigs contain genes or are mostly composed of repeats, we ran the sameBUSCO analysis as above and the redmask.py v0.0.2 command in Red with default pa-rameters to identify repetitive regions [60]. Additionally, we used the Illumina WGS datato calculate kmer completeness and QV score with Merqury [46], as well as to estimategenome-wide heterozygosity with GenomeScope [61]. In brief, we removed adapters andkept only the paired reads from the raw Illumina data using Trimmomatic v0.39 withparameters ILLUMINACLIP: TruSeq3-PE.fa:2:30:10:2:True SLIDINGWINDOW:4:15 LEAD-ING:3 TRAILING:3 MINLEN:36 [62]. Then, for quality assessment, the meryl databasewas built using the trimmed reads with k = 19, based on which Merqury generated thekmer plots to obtain kmer completeness and QV score (Figure S1). Finally, to obtain agenome-wide heterozygosity estimate, we used Jellyfish v2.3.1 [63] to plot a kmer frequencyhistogram from which we calculated the % heterozygosity using GenomeScope v1.0 withk = 21 (as recommended for most species), ensuring that the model fits with >90% accuracy.3.3. Genome Annotation and VisualizationWe performed annotation of our stinging nettle genome assembly using BRAKER3,which allows integration of RNAseq data to support the annotation process and has shownsuperior benchmarking performance in published studies [64–66]. To run the pipeline, wefirst soft-masked the repetitive regions in the genome using the redmask.py v0.0.2 commandin Red, with default parameters [60]. RNAseq data from three different tissue types—leaf,fiber, callus—was retrieved from [24]; raw paired-end Illumina reads were filtered withTrimmomatic v0.39 with parameters ILLUMINACLIP: TruSeq3-PE.fa:2:30:10:2:True SLIDING-WINDOW:4:15 LEADING:3 TRAILING:3 MINLEN:36 [62]. Filtered read pairs were aligned tothe soft-masked genome using Hisat2 v2.2.1 (parameters --dta added), and the alignment filewas converted to BAM format using samtools [67]. Prior to moving forward, the alignmentscore of the RNAseq to the genome was checked to be above 80% on average to confirmthat the individuals from which the RNAseq was obtained from were sufficiently similar toour U. dioica assembly. The resulting BAM file, as well as the Viridiplantae protein databasefrom OrthoDB v11 [68,69], were incorporated in the BRAKER3 pipeline (using --bam and--prot_seq mode). Completeness of the annotation was assessed with BUSCO with --modeprotein (dataset: eudicot_odb10 [58,59]).For transposable elements (TE) annotation, we used the extensive de-novo TE annotator(EDTA) pipeline v.2.2.0 [70] with default parameters. Additionally, we used RepeatOB-server v1 [26] to analyze patterns of tandem repeats across the chromosomes and identifyputative telomeric and centromeric repeats. The location of the putative centromere waspredicted in RepeatOBserver using the default values for Shannon diversity standarddeviation cut-off (Shannon_bin_size = 500, Shannon_SD = 2). Genome-wide patterns ofgene and TE density, Shannon diversity and rearrangements between haplotypes, as wellas putative centromere locations, were visualized using Circos v0.69-8 [71]; computedShannon diversity scores were averaged over a 250 kbp window into a line plot, and thegene and TE annotations were formatted to histograms of counts per 500 kbp window. Toensure proper visualization of TE patterns across the genome, we set the maximum y-axisfor the TE density track to 4000, resulting in the values for six outlier windows with veryhigh TE counts (>4000) being cut off the plot. Location and TE counts in those regions isreported in Table S7. Additionally, we used the LTR Assembly Index (LAI), calculated withLTR_retriever, to assess LTR integrity [25]. LAI is an independent assembly metrics thatPlants 2025, 14, 124 13 of 18can provide useful information regarding how intact the LTRs are, which can be affected byeither age of the LTR or by misassemblies. According to [25], LAIs of 10–20 are considered“Reference” level.3.4. Comparative GenomicsTo investigate the patterns of chromosome evolution within the nettle family, wefirst compiled a list of reference genomes published within the Urticaceae. The quality ofthese genome assemblies was assessed based on the annotation BUSCO score (≥90%). Weincluded Urtica urens L., Parietaria judaica L. (both primary haplotypes from the DarwinTree of Life Project Consortium [1]), and a wild accession of Boehmeria nivea [29]. Foreach genome, the gene annotation file, in gff format, was downloaded together withthe associated protein sequences in amino acid fasta format. Then, the annotation wasconverted into a bed format using the lines corresponding to the “mRNA” annotation,with an extra column matching the header of the protein fasta sequence file. This bed filethen indicates the genome position of every protein. The file conversion was performedmanually with a custom shell script, as needed. For this analysis, we used the H1 haplotypeof our U. dioica assembly since it had a longer total assembly size (quality scores areotherwise similar between the two haplotypes). Once the input was prepared for all thedownloaded datasets and our H1 genome assembly, we ran GENESPACE v1.3.1 [72] tocompare chromosome synteny across the four Urticaceae species. GENESPACE was runwith default parameters. In brief, the initial orthogroups were discovered and identifiedwith OrthoFinder v2.5.4 [73]. Using the synteny information analyzed with MCScanX [74],syntenic blocks based on the gene orders were visualized.3.5. Identification of Sting GenesTo identify putative neurotoxic peptides in our stinging nettle genome, we obtainedthe amino acid sequence of the 4.3-kDa urthionin (∆-Uf1a) and several versions of 6.7-kDaneurotoxic urticatoxin (β/δ-Uf2a, β/δ-Uf2b, β/δ-Dm2a, β/δ-De2a) peptide, described inUrtica ferox G. Forst, Dendrocnide moroides (Wedd.) Chew and D. excelsa (Wedd.) Chew [11].Xie et al. [11] described the above toxins in other Urtica species, including from transcrip-tome data of U. dioica and U. incisa Poir.; however, since these putative toxin transcripts werefound by homology to U. ferox sequences, we omitted them from our analysis. Additionally,we included in our search the two 4-kDa gympietides (Moroidotoxin A, Excelsatoxin A) de-scribed in D. moroides and D. excelsa [12]. After compiling this list of seven sting-associatedneurotoxic peptides found in the literature, we looked for possible homologs in stingingnettle by aligning their protein and transcript sequences to our genome assembly usingminiprot v0.13 (r248) with the parameter -Iut16, which accounted for a flexible size rangeof introns in the homolog detection on the query genome [75]. The visualized alignmentresults of these analyses are reported in Figure S5.4. ConclusionsWe have generated a highly complete, diploid genome assembly of stinging nettle, amulti-purpose species whose use has been interwoven in various cultures for thousandsof years and is seeing renewed interest as a source of natural, sustainable fiber. Despiteits compact size, we found the stinging nettle genome to be highly repetitive, with almosttwo-thirds of its sequence being composed of transposable elements. We also identifysurprisingly high levels of structural variation between haplotypes in the individual wesequenced. While, despite our best efforts, we cannot completely exclude that these aredue to haplotype misassemblies, it seems likely that these two observations are linked,given that transposable elements are known to facilitate the generation of chromosomalPlants 2025, 14, 124 14 of 18rearrangements [76]. Further complexity in the nettle genome is added by the possiblepresence of several polycentric chromosomes, as suggested by diffuse patterns of shorttandem repeats. The stinging nettle genome represents, therefore, an attractive case studyto help understand the evolution of chromosomal variation within and between species.In future studies, it will be interesting to determine whether the inversions we identifiedplay a role in ecotypic adaptation in nettle, as has been observed in other plant species [77].This assembly also provides a valuable resource for the nettle community, as it will greatlyfacilitate analyses of the genetic makeup of useful and interesting traits in the nettle family,such as studies of the genes controlling fiber quality, the production of bioactive moleculesfound in this species, or the formation of nettle’s characteristic stinging hairs.Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/plants14010124/s1, Figure S1: Kmer plot for the U. dioica ssp.dioica assembly; Figure S2: Alignment between the U. dioica assembly from Darwin Tree of Life (DToL)and the U. dioica ssp. dioica H1 and H2 assembly from this study; Figure S3: SyRI alignment betweenU. dioica ssp. dioica H1 and H2 haplotypes; Figure S4: Fourier transformed repeat spectra for eachchromosome; Figure S5: Confirmation of the putative stinging peptide genes; Table S1: Availablegenome assemblies in the Urticaceae family; Table S2: Statistics for haplotype assemblies produced byHifiasm; Table S3: Round 3 and 4 of the manual curation of putative chromosomal inversions basedon Hi-C data; Table S4: Manual curation of putative chromosomal inversions based on long-readsalignment; Table S5: Comparison between H1 and H2 haplotypes using SyRI; Table S6: Annotationstatistics for the two haplotypes; Table S7: Transposable elements annotation statistics; Table S8:Coordinates of putative centromeric regions; Supplementary Methods: Hi-C Protocol.Author Contributions: Conceptualization: Q.C. and M.K.D.; funding and resources: Q.C., M.K.D.,G.G., D.M.P. and M.T.; data production: K.H., C.R.D., M.K. and D.M.P.; formal analyses, investigation,and visualization, K.H. and M.T.; sample preparation and laboratory work: K.H., C.R.D., M.K. andD.M.P.; writing, review, and editing: all authors. All authors have read and agreed to the publishedversion of the manuscript.Funding: This research was supported by NSERC Discovery Grants to MT (RGPIN-2023-03344), QC(RGPIN-2019-04041), and MD (RGPIN-2020-05147).Data Availability Statement: The raw sequencing data and the assembled genome are availableat NCBI under BioProject number PRJNA663211 (haplotype 1) and PRJNA1198346 (haplotype 2).Genome annotation files for genes and TEs are found in Figshare: https://figshare.com/projects/A_high-quality_phased_genome_assembly_of_stinging_nettle_Urtica_dioica_ssp_dioica/230981. Allthe scripts used in this study are available at https://github.com/kaede0e/stinging_nettle_genome_assembly.git. Clones of individual 11-4 are available upon request (quentin.cronk@ubc.ca).Acknowledgments: We thank Eric Gonzales Segovia and Cassandra Elphinstone for their helpwith genome scaffolding and tandem repeats analyses, respectively, as well as Andy Johnson andCassandra Elphinstone for assistance with the flow cytometry. We also gratefully acknowledge theDigital Research Alliance of Canada (DRAC) for access to their computational resources and theNatural History Museum, London (UK) for funding the fieldwork in Europe, undertaken by DPand QC.Conflicts of Interest: The authors declare no conflicts of interest.References1. Taylor, K. Biological Flora of the British Isles: Urtica dioica L. J. Ecol. 2009, 97, 1436–1458. [CrossRef]2. Rejlová, L.; Chrtek, J.; Trávnícˇek, P.; Lucˇanová, M.; Vít, P.; Urfus, T. Polyploid Evolution: The Ultimate Way to Grasp the Nettle.PLoS ONE 2019, 14, e0218389. [CrossRef]3. Harwood, J.; Edom, G. 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genome assembly
Urtica dioica
stinging nettle
trichomes
fiber crops
chromosomal structural variation
Hi-C protocol
A High-Quality Phased Genome Assembly of Stinging Nettle (Urtica dioica ssp. dioica)
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