Abstract
The narrow-leaf bur-reed (Sparganium angustifolium Michx.), a member of the Typhaceae family, is an ecologically important species in temperate Northern Hemisphere aquatic ecosystems. As a phylogenetically basal taxon within the order Poales, S. angustifolium is critical for understanding evolutionary adaptations to aquatic environments. While molecular mechanisms of aquatic adaptation have been extensively studied in Alismatales, genomic resources for basal Poales remain scarce. Here, we present the first chromosome-level genome assembly of S. angustifolium, comprising 486.52 Mb with 99.94% coverage across 15 chromosomes. The assembly demonstrates high continuity, with both contig and scaffold N50 values reaching 33.25 Mb. Genome annotation identified 23,767 protein-coding genes and revealed that repetitive sequences represent 70.16% of the genome. Our findings provide valuable genomic resources for comparative studies of plant adaptation to aquatic environments across different evolutionary lineages.
Subject terms: Data publication and archiving, Molecular ecology
Background & Summary
Narrow-leaf bur-reed (Sparganium angustifolium Michx.), a member of the Typhaceae family, is an ecologically significant species in temperate aquatic ecosystems across the Northern Hemisphere1,2. It occupies diverse habitats including lakes, pools, ditches and streams. This species often forms dense local clones with floating leaves occupying substantial portions of the water surface, spreading clonally via stolons. The plant provides shelter for aquatic animals and its fruits and leaves constitute a valuable food resource for wildfowl2. S. angustifolium is characterized by its slender growth form, typically bearing submerged or floating vegetative structures2. Consequently, its genomic resources are vital for exploring the molecular mechanisms underlying plant aquatic adaptation. Current research on such mechanisms has primarily focused on the order Alismatales3,4. As a phylogenetically basal lineage within the order Poales5, S. angustifolium represents a critical system for understanding the evolutionary adaptation to aquatic environments. Most species in the Typhaceae are emergent, and several genomes of such species, S. stoloniferum6, Typha latifolia7, and T. angustifolia8, have been sequenced. Unlike these emergent species, which grow along water margins with most of their vegetative structures above water, the floating-leaved S. angustifolium inhabits deeper waters, with the majority of its vegetative biomass submerged, thus necessitating distinct aquatic adaptive strategies. Therefore, assembling a high-quality genome of S. angustifolium will provide essential genomic resources for elucidating the mechanism of aquatic adaptation within this key family and across the Poales.
Here we integrated Pacific Biosciences (PacBio) high-fidelity (HiFi) sequencing and high-throughput chromosome conformation capture (Hi-C) technology to assemble a chromosome-level genome of S. angustifolium. The assemble genome size is 486.52 Mb, with a contig N50 length of 33.25 Mb. We predicted 23,767 protein-coding genes and identified repetitive sequences representing 70.16% of the genome (Table 1). This high-quality genome serves as a key resource for investigating genome evolution and exploring aquatic adaptation mechanism within the Poales.
Table 1.
Features of the genome assembly of Sparganium angustifolium.
| Genomic features | Statistics |
|---|---|
| Genome size (Mb) | 486.52 |
| Number of chromosomes | 15 |
| Number of scaffolds | 18 |
| Scaffold N50 size (Mb) | 33.25 |
| Chromosomes percent (%) | 99.94 |
| Number of contigs | 20 |
| Contig Max (Mb) | 47.8 |
| Contig N50 size (Mb) | 33.25 |
| GC content (%) | 42.43 |
| Repetitive sequences (%) | 70.16 |
| Number of protein-coding genes | 23,767 |
| Average gene length (bp) | 5,656 |
| Average CDS length (bp) | 1,440 |
| Average exon length (bp) | 306 |
Methods
Sample collection and genome sequencing
The sequencing sample of S. angustifolium was collected from a river near Genhe City, Inner Mongolia, China (50.7712°N, 121.5027°E). A specimen was deposited at the herbarium of Wuhan University (contact person: Xinwei Xu, xuxw@whu.edu.cn) under the voucher number NMG20240805001. Genomic DNA was extracted using the DNAsecure Plant Kit (Cat. No. DP320-03, Tiangen Biotech). After verification using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific), a Qubit fluorometer (Invitrogen), and gel electrophoresis, the high-quality genomic DNA was used for library construction. The next-generation sequencing (NGS) library was constructed using the NEBNext Ultra II DNA Library Prep Kit according to the manufacturer’s instructions. The Hi-C library was prepared from cross-linked chromatins using Belton et al.’s protocol9. Both the NGS and Hi-C libraries were sequenced on the Illumina NovaSeq 6000 platform to generate 150 bp paired-end reads. Raw reads were subjected to rigorous quality control with the following criteria: (1) reads containing more than 3 ambiguous bases (Ns) in either read of a pair were discarded; (2) read pairs were removed if either read had ≥ 20% of bases with a quality score ≤ 5; and (3) adapter-contaminated read pairs were excluded. After filtering, a total of 31.68 Gb of NGS data and 90.56 Gb of Hi-C data were retained. In addition, TruSeq mRNA-seq libraries were prepared from root, stem, and leaf tissues and sequenced via NGS, yielding 8.65 Gb, 8.51 Gb, and 8.09 Gb of RNA-seq data, respectively. For PacBio HiFi sequencing, a standard SMRT bell library was constructed using the SMRTbell Express Template Prep Kit 2.0 according to the manufacturer’s instructions. Sequencing was then performed on the PacBio Sequel IIe platform, generating 53.27 Gb of raw data.
Genome assembly
The genome size and heterozygosity rate of S. angustifolium was estimated using K-mer frequency analysis implemented in Jellyfish v1.1.1110 based on NGS reads, revealing a genome size of 524.25 Mb and a heterozygosity rate of 0.45% (Fig. 1a). HiFi reads were initially assembled using Hifiasm v0.19.511,12 with default parameters, yielding 576.30 Mb of contigs with a contig N50 of 30.64 Mb. To eliminate heterozygous fragments, reads were mapped back to the assembly using Minimap2 v2.2613, followed by Purge_dups v1.2.514. Contigs were filtered based on NT library alignment, and those with average coverage < 10 × and GC content outside the range of 25.00%–54.00% were discarded using BWA-MEM v0.7.1715. This purification and filtering process finally yielded a contig N50 of 33.25 Mb. The draft genome was scaffolded using Hi-C data, a technology that improves assembly quality by capturing the three-dimensional spatial proximity information of chromatin16. Specifically, HiC-Pro v2.11.417 was used for alignment and 3D-DNA v18092218 for assembly, followed by manual curation in JuiceBox v1.11.0819 to correct misassemblies. This process resulted in a final genome size of 486.52 Mb, with 99.94% (486.23 Mb) anchored onto 15 chromosomes and a scaffold N50 of 33.25 Mb (Table 1). To enhance the presentation of the S. angustifolium genome assembly, we generated a comprehensive circular genome map using Circos v0.6920 (Fig. 1b). This visualization provides an intuitive overview of the genomic architecture, which facilitates further analysis of the S. angustifolium genome. The number of contigs and scaffolds contained in each chromosome, along with the chromosome length and the number of gaps, were presented in Table 2 (Table 2).
Fig. 1.
Overview of the genomic landscape of Sparganium angustifolium. (a) Genome survey of S. angustifolium using k-mer frequency analysis. (b) Circle genomic landscape of S. angstifolium. The circles from the outside to the inside represent Chromosomes length, gene count, repeat sequence content, GC content, gene density, and the picture of S. angustifolium, respectively.
Table 2.
Chromosome characteristics of Sparganium angustifolium.
| Chromosome | Contig Number | Scaffold Number | Chromosome Length(bp) | Gap Number |
|---|---|---|---|---|
| Chr01 | 1 | 2 | 47964942 | 1 |
| Chr02 | 1 | 1 | 44858736 | 0 |
| Chr03 | 1 | 1 | 44083491 | 0 |
| Chr04 | 1 | 1 | 42433007 | 0 |
| Chr05 | 1 | 1 | 33259253 | 0 |
| Chr06 | 1 | 1 | 33245195 | 0 |
| Chr07 | 1 | 1 | 32931560 | 0 |
| Chr08 | 1 | 1 | 30635978 | 0 |
| Chr09 | 1 | 1 | 29220466 | 0 |
| Chr10 | 2 | 1 | 27476996 | 0 |
| Chr11 | 1 | 1 | 26570386 | 0 |
| Chr12 | 1 | 2 | 25935100 | 1 |
| Chr13 | 1 | 1 | 24512531 | 0 |
| Chr14 | 1 | 1 | 22703289 | 0 |
| Chr15 | 2 | 1 | 20665378 | 0 |
Repeat sequence annotation
We comprehensively characterized repetitive sequences in the S. angustifolium genome using both homology-based and de novo approaches. Initial screening with RepeatMasker v4.1.121 against the RepBase database22 was complemented by de novo identification using RepeatModeler v2.0.2a23, collectively revealing 791,281 repetitive elements spanning 341.38 Mb (70.16% of the genome). Long terminal repeat (LTR) retrotransposons dominated the repeat landscape, occupying 215.56 Mb (44.31% of the genome) and representing 63.1% of all repetitive content (Table 3).
Table 3.
General statistics of repetitive sequences in the Sparganium angustifolium genome.
| Class | Superfamily | Classification | Number | Length (bp) | Percentage in the genome (%) |
|---|---|---|---|---|---|
| ClassI (Retrotransposon) | LTR | LTR | 190,758 | 101,166,754 | 20.79 |
| LTR/Cassandra | 2,002 | 57,601 | 0.01 | ||
| LTR/Caulimovirus | 4 | 1,398 | 0.00 | ||
| LTR/Copia | 44,455 | 22,620,562 | 4.65 | ||
| LTR/Gypsy | 128,802 | 91,714,751 | 18.85 | ||
| Non-LTR | LINE/L1 | 41,395 | 31,071,705 | 6.39 | |
| LINE/L1-Tx1 | 107 | 13,422 | 0.00 | ||
| LINE/RTE-BovB | 521 | 79,823 | 0.02 | ||
| Retroposon/L1-derived | 2 | 142 | 0.00 | ||
| SINE/tRNA-RTE | 5 | 231 | 0.00 | ||
| ClassII(DNA_Transposon) | DNA-TE | DNA | 86,309 | 24,675,517 | 5.07 |
| DNA/CMC-EnSpm | 8,111 | 700,980 | 0.14 | ||
| DNA/MULE-MuDR | 17,016 | 7,431,559 | 1.53 | ||
| DNA/MuDR | 153 | 123,849 | 0.03 | ||
| DNA/PIF-Harbinger | 7,114 | 4,455,086 | 0.92 | ||
| DNA/TcMar-Stowaway | 655 | 25,716 | 0.01 | ||
| DNA/Zisupton | 152 | 45,109 | 0.01 | ||
| DNA/hAT-Ac | 5,865 | 2,461,585 | 0.51 | ||
| DNA/hAT-Tag1 | 4,561 | 2,292,724 | 0.47 | ||
| DNA/hAT-Tip100 | 2,189 | 854,600 | 0.18 | ||
| Helitron | RC/Helitron | 4,207 | 882,751 | 0.18 | |
| Others | DNAauto/PILETAA | DNAauto/PILETAA | 235 | 81,891 | 0.02 |
| Satellite | Satellite | 166 | 82,174 | 0.02 | |
| Satellite/centr | Satellite/centr | 28 | 2,186 | 0.00 | |
| Satellite/subtelo | Satellite/subtelo | 1 | 48 | 0.00 | |
| Simple_repeat | Simple_repeat | 37 | 1,299 | 0.00 | |
| Unknown | Unknown | 246,431 | 50,533,737 | 10.39 | |
| Total | 791,281 | 341,377,200 | 70.16 |
Gene prediction and functional annotation
We performed comprehensive gene annotation through transcriptomic and computational approaches. Total RNA was extracted from root, stem, and leaf tissues of S. angustifolium and sequenced as 150 bp paired-end reads on Illumina NovaSeq 6000 platform following library preparation and quality control. RNA-Seq reads were aligned to the genome using HISAT2 v2.2.124, with transcript assembly performed by Cufflinks v2.2.125,26. Gene prediction combined multiple approaches: de novo prediction using AUGUSTUS v3.5.027, SNAP v6.028, GlimmerHMM v3.0.429, and GeneMark-ET v4.3230; homology-based prediction with GeMoMa v1.931,32 against four Poales reference genomes: Sparganium stoloniferum33, Typha angustifolia34, Ananas comosus35, and Tillandsia fasciculata36; and UTR/alternative splicing annotation via PASA v2.5.237. This integrated analysis identified 23,767 protein-coding genes with an average length of 5,656 bp. Functional annotation was performed against GO, KEGG, SWISS-PROT, NR, and eggNOG databases using BLAST v2.2.2538, Interproscan v5.59-91.039, eggnog-mapper v2.1.1240,41, and KOBAS v3.042, with an E-value cutoff of ≤1e-5. Overall, 22,907 genes (96.38%) were successfully annotated (Table 4), including 6,109 genes annotated by all five databases (Fig. 2). The NR database provided the most unique annotations (1,339 genes, Fig. 2). In addition, we used tRNAscan-SE v2.0.1143 to predict tRNAs and Infernal v1.1.444 to identify other types of non-coding RNAs (ncRNAs) based on the Rfam database. This analysis revealed 3,332 ncRNA genes spanning 725,610 bp of genomic sequence. The ncRNA repertoire was dominated by rRNAs (2,403 rRNAs totaling 634,680 bp), 397 tRNAs, 279 snoRNAs, 172 miRNAs, and 81 snRNAs (Table 5).
Table 4.
The annotated genes of the Sparganium angustifolium genome by different databases.
| Database | Number | Percentage (%) | |
|---|---|---|---|
| Total | Protein-coding genes | 23,767 | 100 |
| Annotated | NR | 22,869 | 96.22 |
| SwissProt | 18,050 | 75.95 | |
| KEGG | 9,989 | 42.03 | |
| GO | 10,665 | 44.87 | |
| EggNOG | 21,376 | 89.94 | |
| Not Annotated | 860 | 3.62 | |
| Total annotated | 22,907 | 96.38 |
Fig. 2.
Venn diagram of the number of genes annotated in different public databases.
Table 5.
Number and length of predicted non-coding RNAs.
| Class | Number | Total length (bp) |
|---|---|---|
| rRNA | 2,403 | 634,680 |
| miRNA | 172 | 20,215 |
| tRNA | 397 | 29,734 |
| snRNA | 81 | 11,940 |
| snoRNA | 279 | 29,041 |
| Total | 3,332 | 725,610 |
Data Records
The raw sequence data reported in this paper have been deposited in the Genome Sequence Archive in National Genomics Data Center (NGDC), China National Center for Bioinformation / Beijing Institute of Genomics, Chinese Academy of Sciences. The accession number of these data is CRA02702145 under BioSample SAM143465 and BioProject PRJCA041909. The survey reads (accession numbers ERR1584496546 and ERR1584497147), HiFi reads (accession numbers ERR1583863148, ERR1583853649, and ERR1583853150), and Hi-C reads (accession numbers ERR1584210551 and ERR1584497352) have been deposited in the European Nucleotide Archive. The genome assembly data have been deposited in the Genome Warehouse (GWH) of the National Genomics Data Center (NGDC) with the accession number GWHGEEK00000000.153 and in the European Nucleotide Archive with the accession number GCA_97706350554. The annotation files of genome assembly are available on the Figshare database55.
Technical Validation
Following genome assembly, next-generation sequencing data were aligned to the final assembly using BWA-MEM v0.7.17 with default parameters, yielding an alignment rate of 91.19%. This high alignment rate indicates excellent genome assembly completeness. Subsequently, genome coverage was calculated using mosdepth v0.3.156, revealing an extremely high coverage rate of 99.97%, further supporting the accuracy of genome assembly.
The resulting chromosome-level genome assembly of Sparganium angustifolium spans 486.52 Mb, with both contig N50 and scaffold N50 values at 33.25 Mb. The assembled genome size is notably less than the K-mer estimate (524.25 Mb), a discrepancy likely attributable to heterozygosity or repetitive sequences inflating the k-mer prediction. Assessment of genome completeness using the Liliopsida_odb12 database in BUSCO v5.8.3 showed BUSCO completeness scores of 96.4% (genome) and 97.0% (protein) (Fig. 3a). To further validate assembly accuracy, Hi-C interaction matrices were generated using Samblaster v0.1.2657 and HapHic v1.0.758. The resulting heatmap displayed distinct clustering pattern, with strong interactions between adjacent regions and weaker interactions elsewhere (Fig. 3b), indicative of high-quality chromosome-level assembly.
Fig. 3.
Genome assembly quality assessment. (a) BUSCO assessment of genetic integrity of Sparganium angustifolium. (b) Hi-C interaction heat map of S. angustifolium.
Acknowledgements
This study was supported by the National Water Pollution Control and Treatment Science and Technology Major Project, China (No. 2015ZX07503005). The calculations in this paper were performed using the supercomputing system at the Supercomputing Center of Wuhan University.
Author contributions
X.X. designed the research; J.X. carried out the field collections; X.S. carried out the experiments and performed the data analysis; X.S., J.X. and X.X. wrote and revised the manuscript. All authors read and approved the manuscript.
Data availability
The raw sequence data have been deposited in the Genome Sequence Archive at the National Genomics Data Center under accession number CRA02702145. The survey, HiFi, and Hi-C reads have been deposited in the European Nucleotide Archive under accession numbers ERR1584496546 and ERR1584497147 (survey reads); ERR1583863148, ERR1583853649, and ERR1583853150 (HiFi reads); and ERR1584210551 and ERR1584497352 (Hi-C reads). The genome assembly has been deposited in the Genome Warehouse under accession number GWHGEEK00000000.153 and in the European Nucleotide Archive under accession number GCA_97706350554. The genome annotation files are available in the Figshare database(10.6084/m9.figshare.29375852)55.
Code availability
All bioinformatics tools and software used for genome assembly, annotation, and data analysis in this study were operated strictly according to their official user manuals, with no custom code employed. Software versions and parameters are comprehensively documented in the Methods section.
Competing interests
The authors declare no competing interests.
Footnotes
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Citations
- European Nucleotide Archivehttps://identifiers.org/insdc.sra:ERR15844965 (2025).
- European Nucleotide Archivehttps://identifiers.org/insdc.sra:ERR15844971 (2025).
- European Nucleotide Archivehttps://identifiers.org/insdc.sra:ERR15838631 (2025).
- European Nucleotide Archivehttps://identifiers.org/insdc.sra:ERR15838536 (2025).
- European Nucleotide Archivehttps://identifiers.org/insdc.sra:ERR15838531 (2025).
- European Nucleotide Archivehttps://identifiers.org/insdc.sra:ERR15842105 (2025).
- European Nucleotide Archivehttps://identifiers.org/insdc.sra:ERR15844973 (2025).
- European Nucleotide Archivehttps://identifiers.org/insdc.gca:GCA_977063505 (2025).
Data Availability Statement
The raw sequence data have been deposited in the Genome Sequence Archive at the National Genomics Data Center under accession number CRA02702145. The survey, HiFi, and Hi-C reads have been deposited in the European Nucleotide Archive under accession numbers ERR1584496546 and ERR1584497147 (survey reads); ERR1583863148, ERR1583853649, and ERR1583853150 (HiFi reads); and ERR1584210551 and ERR1584497352 (Hi-C reads). The genome assembly has been deposited in the Genome Warehouse under accession number GWHGEEK00000000.153 and in the European Nucleotide Archive under accession number GCA_97706350554. The genome annotation files are available in the Figshare database(10.6084/m9.figshare.29375852)55.
All bioinformatics tools and software used for genome assembly, annotation, and data analysis in this study were operated strictly according to their official user manuals, with no custom code employed. Software versions and parameters are comprehensively documented in the Methods section.