Chromosome-level genome assembly of helwingia omeiensis: the first genome in the family helwingiaceae

ABSTRACT _Helwingia_, a shrub of the monotypic cosmopolitan family Helwingiaceae, is distinguished by its inflorescence, in which flowers are borne on the midrib of the leaf—a trait not

commonly observed in related plant families. Previous studies have investigated the development of this unusual structure using comparative anatomical methods. However, the scarcity of

genomic data has hindered our understanding of the origins and evolutionary history of this uncommon trait at the molecular level. Here, we report the first high-quality genome of the family

Helwingiaceae. Assembled using HiFi sequencing and Hi-C technologies, the genome of _H. omeiensis_ is anchored to 19 chromosomes, with a total length of 2.75 Gb and a contig N50 length of

6.78 Mb. The BUSCO completeness score of the assembled genome was 98.2%. 53,951 genes were identified, of which 99.7% were annotated in at least one protein database. The high-quality

reference genome of _H. omeiensis_ provides an essential genetic resource and sheds light on the phylogeny and evolution of specific traits in the family Helwingiaceae. SIMILAR CONTENT BEING

VIEWED BY OTHERS CHROMOSOME-LEVEL ASSEMBLIES OF THE ENDEMIC KOREAN SPECIES _ABELIOPHYLLUM DISTICHUM_ AND _FORSYTHIA OVATA_ Article Open access 18 December 2024 CHROMOSOME-LEVEL GENOME

ASSEMBLY OF THE WESTERN FLOWER THRIPS _FRANKLINIELLA OCCIDENTALIS_ Article Open access 04 June 2024 CHROMOSOME-SCALE GENOME ASSEMBLY AND ANNOTATION OF HUZHANG (_REYNOUTRIA JAPONICA_) Article

Open access 21 March 2025 BACKGROUND & SUMMARY Helwingiaceae is a monotypic family in the order Aquifoliales, comprising a single genus _Helwingia_. The innovative structure of this

genus is that the flowers are borne on the midrib of the leaf, which is known as an “epiphyllous inflorescence”, setting them apart from other plants. In addition, the pith, leaves, and

fruits of plants in this genus are traditionally used in herbal medicine to treat dysentery and as diuretic and anti-inflammatory remedies1. The genus includes four species, _H. chinensis_,

_H. himalaica_, _H. japonica_, and _H. omeiensis_, which are all dioecious shrubs mainly found in eastern Asia2,3. Specifically, _H. omeiensis_ is indigenous to Southwest China, and thrives

in moist woodlands and on mountain slopes2. Previous comparative anatomical studies suggested that changes in the position of flower primordium initiation and intercalary growth may

contribute to the formation of this distinct structure4,5,6. With the development of high-throughput sequencing technologies, the genomes of three closely related species in the genus _Ilex_

of the family Aquifoliaceae have been published7,8. However, despite the fact that RNA-seq data and the complete chloroplast genomes of three _Helwingia_ species have been released4,9,10, a

lack of genomic data remains a barrier to studying the evolutionary origin of the family. In this study, we leveraged a combination of short reads, high-fidelity (HiFi) reads, and

chromosome conformation capture (Hi-C) sequencing data to construct a chromosome-level genome assembly for _H. omeiensis_, providing the first genome resource for the family Helwingiaceae.

The length of the genome assembly was 2.75 Gb, with a scaffold N50 of 127.8 Mb and a contig N50 of 6.78 Mb. We identified 1.98 Gb of repetitive elements, accounting for 72.21% of the

assembled genome, as well as 53,951 protein-coding genes. The genome assembly and annotation of _H. omeiensis_ will provide a critical foundation for exploring the genetic basis underpinning

of this unique inflorescence structure and the phylogenetic relationships within the family Helwingiaceae. METHODS PLANT MATERIALS All of the fresh materials were collected from a female

adult plant of _Helwingia omeiensis_ cultivated in Mount Emei Botanical Garden, Sichuan Province, China (N29°35′40, E103°22′40), and the specimens were kept at the Museum of Sichuan

University. The genomic DNA was extracted from young leaves, whereas RNA was extracted from mature leaves and terminal buds. LIBRARY CONSTRUCTION AND SEQUENCING For short-read sequencing,

the sample was randomly fragmented by an ultrasonic processor (Covaris S220; Woburn, MA, USA) to generate DNA fragments approximately 350 bp in length. The DNA fragments were subsequently

constructed through end repair, the addition of a 3′ A tail and the ligation of adapters. Next, the library was sequenced with a DNBSEQ-G400 (BGI, Wuhan, China). The raw short reads were

filtered by SOAPnuke v1.5.610 to remove adapters and low-quality reads. A total of 87.36 Gb of clean data were obtained for _H. omeiensis_ (Table 1). For HiFi (high-fidelity) sequencing,

high-quality genomic DNA was sheared using Megaruptor® 3 (Diagenode), and subreads with a length of 20 kb were further selected using Sage ELF to prepare the PacBio HiFi libraries in CCS

mode on the Pacific Biosciences Sequel II System (Supplementary Figure S1). Finally, 50.32 Gb of long clean reads were generated (Table 1), with mean lengths of 13.0 kb and 14.5 kb,

respectively. Hi-C technology captures sequence interactions between all DNA segments within chromosomes to obtain information on interactions between segments of the genome for assisted

genome assembly11. Fresh leaves of the same individual were used to construct Hi-C libraries, and the MboI restriction enzyme was used for DNA ligation. After tailing, pulldown, and adapter

ligation, the DNA library was sequenced on an Illumina HiSeq X Ten System (BGI, Wuhan, China) with a strategy of 2 × 150 bp. After filtering low-quality reads, 221.52 Gb of clean Hi-C data

were obtained (Table 1). RNA SEQUENCING Mature leaves and young terminal buds of the same individual were collected for RNA extraction. The RNA-seq library was constructed using the Illumina

standard protocol (San Diego, CA, United States) and sequenced on the Illumina HiSeq X Ten platform (BGI, Wuhan, China). The raw data were filtered by Cutadapt v1.1612 to remove adapters

and low-quality reads. After quality control by FastQC v0.11.8 (https://github.com/s-andrews/FastQC), 10.46 Gb of paired-end short clean reads were generated from the RNA-seq library (Table 

1). GENOME SURVEY AND _DE NOVO_ ASSEMBLY Jellyfish v2.1.413 was used to quickly count _K_-mer frequencies ranging from 17 to 31, and then GenomeScope14 predicted genomic features using a

_K_-mer-based statistical approach (Supplementary Table S1). The _H. omeiensis_ genome was estimated to be 2.54 Gb in size, with a heterozygosity rate of 1.19% and repetitive sequences

accounting for 54.85% of the total length of the genome (Fig. 1). Using 50.32 Gb of clean HiFi reads with hifiasm v0.19.6-r59515, we generated a genome assembly of 2.92 Gb in size with a

contig N50 of 6.21 Mb. Following that, Chromap v0.2.5-r47316 was utilized to align Hi-C clean reads to the contig assembly, and according to the strength of interactions between pairs of

reciprocal sequences, YaHS v1.2a.117 was used to anchor contigs onto 1,584 scaffolds. Next, using Juicebox v1.11.0818, we visualized the Hi-C contact maps of the scaffold assembly and made

final refinements to the genome assembly. With reference to chromosome counts indexed in the Chromosome Counts Database (CCDB)19 (https://ccdb.tau.ac.il/) and the whole-genome Hi-C

interaction heatmap, we identified the 19 longest scaffolds as pseudo-chromosomes (Fig. 2). TGS-GapCloser v1.2.120 filled 75 of the 1,011 gaps in the scaffold assembly based on HiFi reads.

The final assembly had a total length of 2.75 Gb, with a contig N50 of 6.78 Mb. The length of 19 pseudochromosomes was 2.38 Gb, with a maximum chromosome length of 153.79 Mb (Table 2). Since

there is no reference genome for this species, we numbered the chromosomes in order from largest to smallest (Fig. 3 and Table 3). GENE ANNOTATION To perform a comprehensive prediction of

protein-coding genes, the GETA v2.5.6 pipeline (https://github.com/chenlianfu/geta) was used for automatic genome-wide annotation. First, RepeatModeler v2.0.321 and DeepTE22 were used for

self-training and to construct a repeat library. On this basis, RepeatMasker v4.1.2-p123 was employed to predict and combine repetitive elements for homology-based methods. The analysis

revealed that 72.21% of the genome was composed of repetitive sequences, including 46.39% long-terminal repeat (LTR) retrotransposons and 19.43% DNA transposons (Table 4). After masking

repetitive sequences in the genome, three strategies (homology-based, RNA-seq-guided, and _ab initio_ methods) were used for the annotation process. For the RNA-seq-guided method, the RNA

sequencing data were provided to HISAT2 v2.1.024 and SAMtools v1.1125 to map the data to the repeat-masked genome. Then, TransDecoder v5.5.0 (https://github.com/TransDecoder/TransDecoder)

was used to predict the open reading frame (ORF), and filter out the gene models with identities greater than 80% at the amino acid level between pairs to obtain nonredundant results.

Protein sequences from _Vitis vinifera_, _Arabidopsis thaliana_, _Solanum lycopersicum_, _Daucus carota_, and _Ilex latifolia_ were aligned to the query genome as homologous proteins using

GeneWise v2.4.126 to estimate protein-coding genes (Supplementary Table S2). _Ab initio_ prediction was carried out with AUGUSTUS v3.4.027, which guided by previous prediction results. Based

on the GETA pipeline, all the outputs were validated using HMMER v3.3.228 and NCBI-BLAST + v2.13.0 + before being integrated into a complete and nonredundant set of gene annotations.

Following the alignments by DIAMOND v2.0.1529, gene functions were indicated using the Nonredundant Protein Sequence Database (NR)30, InterPro31, UniProt32, and EggNOG33 with an e-value of

1e-5. In addition, GO annotation was performed by KOBAS34 (http://kobas.cbi.pku.edu.cnwas) aligned with the _Arabidopsis thaliana_ database. DATA RECORDS All the raw sequencing reads of _H.

omeiensis_ were uploaded to the NCBI database under accession number SRP43521335. The genome assembly had been submitted to Genome Warehouse in China National Center for Bioinformation under

accession number GWHEQHK0000000036 and European Nucleotide Archive (ENA) with accession number GCA_964187755.237. The annotation files of the genome are available in the figshare database:

https://doi.org/10.6084/m9.figshare.22817414.v338. TECHNICAL VALIDATION EVALUATION OF THE GENOME ASSEMBLY AND ANNOTATION To assess the integrity of the assembly, short reads were mapped to

the genomes using minimap239, giving a mapping rate of 96.59% and a genome coverage of 99.85%. The alignment rate of RNA sequencing reads was 96.95% and 94.10% for two _H. omeiensis_ samples

by HISAT2 v2.1.0 (Supplementary Table S3)24. The completeness and accuracy of the final genome assembly were checked by Benchmarking Universal Single-Copy Orthologs (BUSCO) v5.4.240 with

eudicots_odb10. The results showed that 98.2% of orthologs of eudicots could be identified in the assembly (Supplementary Figure S2). Moreover, the values evaluated by Merqury v1.341 based

on short reads also showed high consensus quality (accuracy > 99.99%, QV > 58) and low base-level error rates (1.37 × 10−6). In addition, the LTR Assembly Index (LAI) score of the

whole-genome assembly was calculated to be 24.52, exceeding that of rice (MSUV7) and _Arabidopsis_ (TAIR10), reaching the ‘gold quality’42. These results demonstrated that the assembly is

reliable and has high base-level accuracy, high completeness, and high contiguity. Via multiple annotation approaches, we identified 53,951 protein-coding genes in the _H. omeiensis_ genome

(Table 5). BUSCO analysis showed the completeness of predicted genes was 94.5% (Supplementary Figure S2). The functional analysis revealed that 99.7% of the protein-encoding genes could be

annotated in at least one of five public databases (Fig. 4). CODE AVAILABILITY (1) SOAPnuke v1.5.6: parameters: -n 0.01 -l 20 -q 0.1 -i -Q 2 -G -M 2 -A 0.5 -d (2) Cutadapt v1.16: parameters:

-a AGATCGGAAG -q 20 All the other software and pipelines not listed or described in the methods section used the default parameters. REFERENCES * Chen Lin, L. W.-j. _et al_. Overview of

Pharmaceutical Research on Helwingia Willd. _Journal of Liaoning University of Traditional Chinese Medicine_ 14, 116–118 (2012). * Wu, R. H. W. Z., Raven, P. H., Hong, D. Y. _Flora of China

(Apiaceae through Ericaceae)_. Vol. 14 (Science Press, 2005). * Miller, C. The World Flora Online – Research Infrastructure for Plant Conservation. _Biodiversity Information Science and

Standards_ (2019). * Sun, C., Yu, G., Bao, M., Zheng, B. & Ning, G. Biological pattern and transcriptomic exploration and phylogenetic analysis in the odd floral architecture tree:

Helwingia willd. _BMC Res Notes_ 7, 402 (2014). Article  PubMed  PubMed Central  Google Scholar  * Ao, C. & Tobe, H. Floral morphology and embryology of Helwingia (Helwingiaceae,

Aquifoliales): systematic and evolutionary implications. _J Plant Res_ 128, 161–175 (2015). Article  PubMed  Google Scholar  * Dickinson, T. A. & Sattler, R. Development of the

epiphyllous inflorescence of helwingia japonica (helwingiaceae). _American Journal of Botany_ 62, 962–973 (1975). Article  Google Scholar  * Yao, X., Lu, Z., Song, Y., Hu, X. & Corlett,

R. T. A chromosome-scale genome assembly for the holly (Ilex polyneura) provides insights into genomic adaptations to elevation in Southwest China. _Hortic Res_ 9 (2022). * Kong, B. L. _et

al_. Chromosomal level genome of Ilex asprella and insight into antiviral triterpenoid pathway. _Genomics_ 114, 110366 (2022). Article  CAS  PubMed  Google Scholar  * Zhang, C. _et al_.

Asterid Phylogenomics/Phylotranscriptomics Uncover Morphological Evolutionary Histories and Support Phylogenetic Placement for Numerous Whole-Genome Duplications. _Mol Biol Evol_ 37,

3188–3210 (2020). Article  CAS  PubMed  Google Scholar  * Chen, Y. _et al_. SOAPnuke: a MapReduce acceleration-supported software for integrated quality control and preprocessing of

high-throughput sequencing data. _Gigascience_ 7, 1–6 (2018). Article  ADS  PubMed  PubMed Central  Google Scholar  * Louwers, M., Splinter, E., van Driel, R., de Laat, W. & Stam, M.

Studying physical chromatin interactions in plants using Chromosome Conformation Capture (3C). _Nat Protoc_ 4, 1216–1229 (2009). Article  CAS  PubMed  Google Scholar  * Martin, M. Cutadapt

Removes Adapter Sequences From High-Throughput Sequencing Reads. _EMBnet.journal_ 17, 10–12. * Marcais, G. & Kingsford, C. A fast, lock-free approach for efficient parallel counting of

occurrences of k-mers. _Bioinformatics_ 27, 764–770 (2011). Article  CAS  PubMed  PubMed Central  Google Scholar  * Vurture, G. W. _et al_. GenomeScope: fast reference-free genome profiling

from short reads. _Bioinformatics_ 33, 2202–2204 (2017). Article  CAS  PubMed  PubMed Central  Google Scholar  * Cheng, H., Concepcion, G. T., Feng, X., Zhang, H. & Li, H.

Haplotype-resolved _de novo_ assembly using phased assembly graphs with hifiasm. _Nat Methods_ 18, 170–175 (2021). Article  CAS  PubMed  PubMed Central  Google Scholar  * Zhang, H. _et al_.

Fast alignment and preprocessing of chromatin profiles with Chromap. _Nature Communications_ 12 (2021). * Zhou, C., McCarthy, S. A., Durbin, R. & Alkan, C. YaHS: yet another Hi-C

scaffolding tool. _Bioinformatics_ 39 (2023). * Durand, N. C. _et al_. Juicebox Provides a Visualization System for Hi-C Contact Maps with Unlimited Zoom. _Cell Syst_ 3, 99–101 (2016).

Article  CAS  PubMed  PubMed Central  Google Scholar  * Rice, A. _et al_. The Chromosome Counts Database (CCDB) – a community resource of plant chromosome numbers. _New Phytologist_ 206,

19–26 (2014). Article  PubMed  Google Scholar  * Xu, M. _et al_. TGS-GapCloser: A fast and accurate gap closer for large genomes with low coverage of error-prone long reads. _GigaScience_ 9

(2020). * Flynn, J. M. _et al_. RepeatModeler2 for automated genomic discovery of transposable element families. _Proc Natl Acad Sci USA_ 117, 9451–9457 (2020). Article  ADS  CAS  PubMed 

PubMed Central  Google Scholar  * Yan, H., Bombarely, A., Li, S. & Valencia, A. DeepTE: a computational method for de novo classification of transposons with convolutional neural

network. _Bioinformatics_ 36, 4269–4275 (2020). Article  CAS  PubMed  Google Scholar  * Tarailo-Graovac, M. & Chen, N. Using RepeatMasker to identify repetitive elements in genomic

sequences. _Curr Protoc Bioinformatics_ CHAPTER 4, 4 10 11–14 10 14 (2009). Google Scholar  * Kim, D., Paggi, J. M., Park, C., Bennett, C. & Salzberg, S. L. Graph-based genome alignment

and genotyping with HISAT2 and HISAT-genotype. _Nat Biotechnol_ 37, 907–915 (2019). Article  CAS  PubMed  PubMed Central  Google Scholar  * Li, H. _et al_. The Sequence Alignment/Map format

and SAMtools. _Bioinformatics_ 25, 2078–2079 (2009). Article  PubMed  PubMed Central  Google Scholar  * Birney, E., Clamp, M. & Durbin, R. GeneWise and Genomewise. _Genome Res_ 14,

988–995 (2004). Article  CAS  PubMed  PubMed Central  Google Scholar  * Stanke, M. _et al_. AUGUSTUS: ab initio prediction of alternative transcripts. _Nucleic Acids Res_ 34, W435–439

(2006). Article  CAS  PubMed  PubMed Central  Google Scholar  * Eddy, S. R. Accelerated Profile HMM Searches. _PLoS Comput Biol_ 7, e1002195 (2011). Article  ADS  MathSciNet  CAS  PubMed 

PubMed Central  Google Scholar  * Buchfink, B., Xie, C. & Huson, D. H. Fast and sensitive protein alignment using DIAMOND. _Nat Methods_ 12, 59–60 (2015). Article  CAS  PubMed  Google

Scholar  * Sayers, E. W. _et al_. Database resources of the National Center for Biotechnology Information in 2023. _Nucleic Acids Research_ 51, D29–D38 (2023). Article  CAS  PubMed  Google

Scholar  * Mitchell, A. _et al_. The InterPro protein families database: the classification resource after 15 years. _Nucleic Acids Res_ 43, D213–221 (2015). Article  PubMed  Google Scholar

  * Boeckmann, B. _et al_. The SWISS-PROT protein knowledgebase and its supplement TrEMBL in 2003. _Nucleic Acids Res_ 31, 365–370 (2003). Article  CAS  PubMed  PubMed Central  Google

Scholar  * Huerta-Cepas, J. _et al_. eggNOG 5.0: a hierarchical, functionally and phylogenetically annotated orthology resource based on 5090 organisms and 2502 viruses. _Nucleic Acids Res_

47, D309–D314 (2019). Article  CAS  PubMed  Google Scholar  * Bu, D. _et al_. KOBAS-i: intelligent prioritization and exploratory visualization of biological functions for gene enrichment

analysis. _Nucleic Acids Research_ 49, W317–W325 (2021). Article  CAS  PubMed  PubMed Central  Google Scholar  * _NCBI Sequence Read Archive_ https://identifiers.org/ncbi/insdc.sra:SRP435213

(2023). * _National Genomics Data Center_ https://ngdc.cncb.ac.cn/gwh/Assembly/83104/show (2023). * _European Nucleotide Archive_ http://identifiers.org/insdc.gca:GCA_964187755.2 (2024). *

Chen, Y. The annotation of _Helwingia omeiensis_ genome assembly. _figshare_ https://doi.org/10.6084/m9.figshare.22817414.v3 (2023). * Li, H. Minimap2: pairwise alignment for nucleotide

sequences. _Bioinformatics_ 34, 3094–3100 (2018). Article  CAS  PubMed  PubMed Central  Google Scholar  * Simao, F. A., Waterhouse, R. M., Ioannidis, P., Kriventseva, E. V. & Zdobnov, E.

M. BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs. _Bioinformatics_ 31, 3210–3212 (2015). Article  CAS  PubMed  Google Scholar  * Rhie, A., Walenz,

B. P., Koren, S. & Phillippy, A. M. Merqury: reference-free quality, completeness, and phasing assessment for genome assemblies. _Genome Biology_ 21 (2020). * Ou, S., Chen, J. &

Jiang, N. Assessing genome assembly quality using the LTR Assembly Index (LAI). _Nucleic Acids Research_ 46, e126–e126 (2018). PubMed  PubMed Central  Google Scholar  Download references

ACKNOWLEDGEMENTS This research was supported by the Natural Science Foundation of China (32171606, 41771055). AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Key Laboratory for Bio-Resource

and Eco-Environment of Ministry of Education & Sichuan Zoige Alpine Wetland Ecosystem National Observation and Research Station, College of Life Science, Sichuan University, Chengdu,

China Yanyu Chen, Landi Feng, Hao Lin, Jianquan Liu & Quanjun Hu Authors * Yanyu Chen View author publications You can also search for this author inPubMed Google Scholar * Landi Feng

View author publications You can also search for this author inPubMed Google Scholar * Hao Lin View author publications You can also search for this author inPubMed Google Scholar * Jianquan

Liu View author publications You can also search for this author inPubMed Google Scholar * Quanjun Hu View author publications You can also search for this author inPubMed Google Scholar

CONTRIBUTIONS Y.C., L.F. and H.L. collected the materials and performed the genome sequencing and assembly. Y.C. performed the data validation and analyses. Y.C., J.L. and Q.H. wrote the

manuscript. All the authors approved the submitted version. CORRESPONDING AUTHOR Correspondence to Quanjun Hu. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing

interests. ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. SUPPLEMENTARY

INFORMATION SUPPLEMENTARY INFORMATION RIGHTS AND PERMISSIONS OPEN ACCESS This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing,

adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons

licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a

credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted

use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Reprints and permissions ABOUT

THIS ARTICLE CITE THIS ARTICLE Chen, Y., Feng, L., Lin, H. _et al._ Chromosome-level genome assembly of _Helwingia omeiensis_: the first genome in the family Helwingiaceae. _Sci Data_ 11,

719 (2024). https://doi.org/10.1038/s41597-024-03568-7 Download citation * Received: 26 December 2023 * Accepted: 25 June 2024 * Published: 02 July 2024 * DOI:

https://doi.org/10.1038/s41597-024-03568-7 SHARE THIS ARTICLE Anyone you share the following link with will be able to read this content: Get shareable link Sorry, a shareable link is not

currently available for this article. Copy to clipboard Provided by the Springer Nature SharedIt content-sharing initiative