
==== Front
Hortic Res
Hortic Res
hr
Horticulture Research
2662-6810
2052-7276
Oxford University Press

10.1093/hr/uhae185
uhae185
Article
AcademicSubjects/SCI01210
AcademicSubjects/SCI01140
The gap-free genome of Forsythia suspensa illuminates the intricate landscape of centromeres
Cui Jian School of Architecture & Built Environment, The University of Adelaide, Adelaide, 5005, Australia

Zhu Congle Key Laboratory of Seed Innovation, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100101, China

Shen Lisha Key Laboratory of Seed Innovation, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100101, China

Yi Congyang Key Laboratory of Seed Innovation, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100101, China

Wu Rong College of Life Science and Technology, Inner Mongolia Normal University, Hohhot, 010022, China

Sun Xiaoyang College of Grassland Science, Qingdao Agricultural University, Qingdao, 266109, China

Han Fangpu Key Laboratory of Seed Innovation, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100101, China

https://orcid.org/0009-0004-0672-9334
Li Yong College of Life Science and Technology, Inner Mongolia Normal University, Hohhot, 010022, China

Liu Yang Key Laboratory of Seed Innovation, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100101, China

Corresponding authors. E-mails: fphan@genetics.ac.cn; 20220053@imnu.edu.cn; yangliu@genetics.ac.cn
These authors contributed equally.

9 2024
10 7 2024
10 7 2024
11 9 uhae18505 5 2024
01 7 2024
01 9 2024
© The Author(s) 2024. Published by Oxford University Press on behalf of Nanjing Agricultural University.
2024
https://creativecommons.org/licenses/by/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Forsythia suspensa, commonly known as weeping forsythia, holds significance in traditional medicine and horticulture. Despite its ecological and cultural importance, the existing reference genome presents challenges with duplications and gaps, hindering in-depth genomic analyses. Here, we present a Telomere-to-Telomere (T2T) assembly of the F. suspensa genome, integrating Oxford Nanopore Technologies (ONT) ultra-long, Hi-C datasets, and high-fidelity (HiFi) sequencing data. The T2T reference genome (Fsus-CHAU) consists of 14 chromosomes, totaling 688.79 Mb, and encompasses 33 932 predicted protein-coding genes. Additionally, we characterize functional centromeres in the F. suspensa genome by developing a specific CENH3 antibody. We demonstrate that centromeric regions in F. suspensa exhibit a diverse array of satellites, showcasing distinctive types with unconventional lengths across various chromosomes. This discovery offers implications for the adaptability of CENH3 and the potential influence on centromere dynamics. Furthermore, after assessing the insertion time of full-length LTRs within centromeric regions, we found that they are older compared to those across the entire genome, contrasting with observations in other species where centromeric retrotransposons are typically young. We hypothesize that asexual reproduction may impact retrotransposon dynamics, influencing centromere evolution. In conclusion, our T2T assembly of the F. suspensa genome, accompanied by detailed genomic annotations and centromere analysis, significantly enhances F. suspensa potential as a subject of study in fields ranging from ecology and horticulture to traditional medicine.

National Natural Science Foundation of China 10.13039/501100001809 32360307 and 32300491
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pmcIntroduction

Forsythia suspensa, commonly known as Forsythia, is a prominent deciduous shrub within the Oleaceae family, typically attaining heights of 1.5 to 3.0 meters. Its early spring bloom, featuring abundant, golden, and fragrant flowers, makes it a highly sought-after ornamental shrub for enhancing landscapes. F. suspensa’s ecological attributes include its ability to retain soil moisture and its remarkable resistance to cold and drought, which has led to its frequent use for erosion control in hilly terrains and along roadsides. Furthermore, F. suspensa holds significant medicinal value, with its fruit extracts being a key component in many traditional Chinese remedies for the common cold. Given its significance in horticulture, ecology, and traditional medicine, Forsythia has gained increasing attention in recent years [1]. The Forsythia genus, belonging to Tribe Forsythieae (Oleaceae), includes 10 recognized species and is diploid, with a chromosome number of 2n = 28. Forsythia likiangensis and Forsythia giraldiana represent the basal lineages, followed by Forsythia europaea. These three species are characterized by minutely serrate or entire leaf margins. The remaining species, primarily distributed in East Asia, form two major clades. One clade includes Forsythia ovata, Forsythia velutina, and Forsythia japonica, which are morphologically supported by broadly ovate leaves. The other clade consists of F. suspensa, Forsythia saxatilis, Forsythia viridissima, and Forsythia koreana, characterized by lanceolate leaves, except for F. suspensa, which has broad ovate leaves [2].

Although a previous study provided a draft genome of weeping forsythia (F. suspensa), it did not achieve assembly at the chromosomal level [3]. Building on this, a recent chromosome-scale genomic assembly of weeping forsythia was completed, with a total of 712.9 Mb genomic sequences organized into 14 pseudo-chromosomes [4]. Despite these advances in F. suspensa genome research, significant challenges remain. The presence of numerous unassembled or unplaced sequences poses a major obstacle to comprehensive genomic investigations. These challenges emphasize the need for further research and improved genome assembly to fully unlock F. suspensa’s potential as a subject of study.

Centromeres serve as crucial genomic structures essential for ensuring the precise segregation of chromosomes during cell division [5]. Two key components are particularly essential in understanding centromere structure and function: CENH3 and centromeric repetitive sequences. CENH3, alternatively referred to as CENP-A in humans, represents a specialized histone H3 variant pivotal for defining centromere identity. Epigenetically labeled, it is uniquely positioned within the centromeric domains of chromosomes. CENH3 assumes a critical function in enlisting additional centromeric proteins and instigating the assembly of the kinetochore, a sophisticated protein complex responsible for interfacing with spindle microtubules during cellular division [6]. Centromeric repetitive sequences, on the other hand, constitute a significant portion of centromere DNA. These repetitive sequences are typically characterized by satellite DNA, which are highly divergent among different species. In plants, centromeres typically encompass numerous copies of simple tandem repeat DNA sequences, organized into extended arrays spanning several megabases in length. These arrays are periodically interrupted by the insertion of long-terminal repeat retrotransposons [7]. The distinctive structural organization of plant centromeres is essential for their proper function and contributes significantly to the precise segregation of chromosomes during cell division [8].

Recent advancements in ultra-long DNA sequencing technologies and the refinement of advanced computational tools have revolutionized the analysis of centromere structure and function. One significant outcome of these advancements is the successful assembly of gap-free reference genomes for several plant species [9–16]. These reference genomes have unveiled the previously concealed complexities and variations within centromeric regions. In Arabidopsis, the processes of satellite homogenization and retrotransposon invasions have been identified as crucial factors in the continuous evolution of centromeres [17]. Furthermore, investigations into Arabidopsis populations have uncovered the existence of centromere diversity and have highlighted the dynamic nature of centromere evolution, driven by rapid cycles of transposon invasion and purging, which are in turn influenced by satellite homogenization [18]. Additionally, during the evolutionary journey of the soybean genome, a remarkable occurrence has been the extensive repositioning of centromeres [19]. This phenomenon of centromere repositioning showcases the genome’s remarkable adaptability in response to evolutionary pressures and genetic changes. It emphasizes the dynamic nature of centromeres and their crucial role in maintaining genome stability. As a result of incomplete assembly in the repetitive regions of previously published F. suspensa reference genomes [4], our understanding of the comprehensive size and structural aspects of centromeres in F. suspensa remains limited.

In this research, we introduced a gap-free genome assembly of F. suspensa through the integration of Oxford Nanopore Technology (ONT) sequencing, PacBio HiFi, and high-throughput chromosome conformation capture (Hi-C) technologies. Our latest assembly, denoted as Fsus-CHAU, surpasses the previously published F. suspensa genome in terms of continuity and completeness. This now enables a comprehensive analysis of centromere architecture and evolution on a global scale.

Results

A comprehensive, gap-free telomere-to-telomere (T2T) reference genome for F. suspensa

F. suspensa was selected for sequencing due to its multifaceted significance in medicine, environmental conservation, and landscape enhancement (Fig. 1B). Through k-mer analysis of Illumina reads, we conducted a thorough assessment of the F. suspensa genome, yielding a k-mer value of 19. This analysis led to an estimated F. suspensa genome size of approximately 734.07 Mb, with a calculated heterozygosity level of 1.47% (Table S1, see online supplementary material).

Figure 1 Summary of the Fsus-CHAU genome and its associated phenotypic characteristics. A Circos plot. Starting from the outermost to the innermost ring: (a) chromosome, (b) gene number density, (c) LTR density, (d) GC content, and (e) syntenic block. B The phenotypes of different tissues, including folwer, fruit, stem, leaf, and the whole plant in Forsythia suspensa. C The karyotype analysis of 14 chromosome pairs in F. suspensa. D Chromatin interaction map generated by Hi-C for the Fsus-CHAU genome.

In order to attain a genome assembly of superior quality for F. suspensa, we embarked on a comprehensive sequencing endeavor by employing a variety of state-of-the-art technologies. Our approach involved the integration of 58 Gb (~81 × coverage) PacBio HiFi reads, 21 Gb (~30 × coverage) of ONT ultra-long reads, and 78 Gb (~109 × coverage) of Hi-C data (Tables S2–S4, see online supplementary material). After obtaining reads from both HiFi and ONT ultra-long sequencing, primary assembly was conducted, resulting in three sets of primary contig genomes: hifiasm-hifi-ont, which was a hybrid assembly using both HiFi and ONT ultra-long sequencing data; hifiasm-hifi, created using only HiFi sequencing data; and nextdenovo-ont, which utilized ONT ultra-long sequencing data with the NextDenovo software. Following a comprehensive evaluation of each assembly version, the contig-level hifiasm-hifi genome with the highest quality was selected as the T2T genome backbone (Table S5, see online supplementary material). Subsequent analyses, such as removal of assembly contaminants, Hi-C integration, assembly correction, telomere repair, gap filling, and other operations were all based on this version. After Hi-C assembly and manual adjustments, a total of 720 688 960 base pairs of contig sequences were mapped to the 14 chromosomes of the hifiasm-hifi assembly version (Table S6, see online supplementary material), consistent with our karyotype analysis of 14 chromosome pairs in F. suspensa (Fig. 1C). However, six gaps remained in the assembly. To address this, we employed ONT ultra-long reads to map onto the initial assembly, filling in these remaining gaps. We found the aligned position spanned across both ends of the six gaps, and thus selected the optimal alignment region from the longest aligned length region for gap filling. This data was then used to replace the sequence in the genome that encompassed the gap region (Fig. S1, see online supplementary material). This comprehensive approach culminated in the creation of a gap-free genome, aptly named Fsus-CHAU. This exceptional genome comprised 14 chromosomes, totaling 688.79 Mb in cumulative length, with an N50 length of 48.48 Mb (Table 1). In the final step of our analysis, we conducted a targeted search using a telomere repeat sequence (5′-CCCTAAA/TTTAGGG-3′). This effort successfully identified 28 telomeres. The upstream copy number of 5′-CCCTAAA ranged from 1117 copies on chromosome 6 to 2363 copies on chromosome 7, and the downstream copy number of 5′-TTTAGGG ranged from 1346 copies on chromosome 7 to 2769 copies on chromosome 9 (Table S7, see online supplementary material). Ultimately, this resulted in the generation of 14 T2T pseudomolecules within the F. suspensa assemblies (Fig. 1A).

Table 1 Global statistics of Fsus-CHAU genome assembly and annotation.

Assembly	Fsus-CHAU	Annotation	Fsus-CHAU	
Genome size (Mb)	688.79	Gene number	33 932	
Number of contigs	155	Genome BUSCO (%)	98.6	
Contig N50 (Mb)	48.48	Average gene length (bp)	5279	
Number of contigs (length ≥2 kb)	14	Number of non-coding RNAs	8398	
Number of gap-free chromosomes	14	TE percentage (%)	62	
Number of telomeres	28	Gypsy family percentage (%)	14	
Number of definite centromeres	14	Copia family percentage (%)	13	

The precision and comprehensiveness of the F. suspensa genome assembly underwent extensive validation through various methods. Initially, the Hi-C heatmap clearly indicated a strong uniformity among all chromosomes, confirming the accurate organization and alignment of contigs in the new assembly (Fig. 1D). Subsequently, the assembly’s accuracy was further bolstered by the observation that over 99% of PacBio HiFi reads were successfully aligned to the new assembly (Table S8, see online supplementary material). Lastly, the completeness of the assembly was evaluated using the Benchmarking Universal Single-Copy Orthologs (BUSCO) analysis, achieving an impressive 98.6% completion rate (1591 out of 1614 BUSCOs) (Table 1; Table S9, see online supplementary material). Our gap-free Fsus-CHAU genome demonstrates superior contiguity and completeness compared to previously reported assembly (Fig. 2A). Specifically, our assembly boasts a contig N50 of 48.48 Mb, significantly higher than the previous weeping forsythia genome’s contig N50 of 7.3 Mb. Previous reference genomes contained hundreds or thousands of gaps [4]. However, our assembly has added 8.91 Mb of new sequences and effectively filled in all existing gaps (Fig. 2A, Table 1).

Figure 2 Collinearity between Fsus-CHAU and weeping forsythia. A Structural variations between the Fsus-CHAU and weeping forsythia genome. B Summary of genomic annotation: the ‘Other’ genomic composition encompasses non-protein-coding genes, unclassified repeat regions, and genomic segments that defy annotation as either genes or repeats. C Proportion of different repeat types in the Fsus-CHAU genome.

Genome annotation

In the process of annotating the gene models of F. suspensa genome, we employed a comprehensive approach that combined multiple prediction methods, homology-based analyses, and transcriptome data. This rigorous annotation pipeline allowed us to achieve a thorough and accurate gene annotation (Fig. S2, see online supplementary material). Our analysis identified a total of 33 932 gene models in the F. suspensa genome (Table 1; Table S10, see online supplementary material). Notably, these gene models captured 98.0% of the BUSCO 1614 reference gene set, further attesting to the quality of our annotation. The average gene length was estimated to be 5279 bp, with an average of 4.77 exons per gene (Table S11, see online supplementary material). Additionally, 20 242 genes were supported by transcriptome data, accounting for 59.65% of the total gene models. In parallel with protein-coding genes, our analysis also predicted 8398 non-coding RNAs within the F. suspensa genome. This encompassed 190 microRNAs, 716 transfer RNAs, 4919 ribosomal RNAs, and 2573 small nuclear RNAs, contributing to a comprehensive and detailed gene annotation (Table 1; Table S12, see online supplementary material).

For a comprehensive understanding of the repetitive landscape in the F. suspensa genome, we leveraged a combination of de novo and homoeology-based methods. Ultimately, we identified that approximately 72% of our gap-free genome is comprised of repetitive sequences (Fig. 2B; Tables S13 and S14, see online supplementary material). Notably, the majority of these repeats were attributed to transposable elements (TEs), encompassing 62% of the genome and totaling 450 664 727 bp in length. The TE category was predominantly characterized by the long terminal repeat (LTR) type, with 14% of these elements belonging to the Gypsy family and 13% to the Copia family (Fig. 2C, Table 1; Table S13, see online supplementary material).

Figure 3 Genomic mapping of functional centromeres in Forsythia suspensa. A Multiple alignments of CENH3 homologs from different plant lineages including Zea mays, Oryza sativa, Arabidopsis thaliana, Glycine max, and F. suspensa. The peptide used to generate the anti-CENH3 antibody, with the N-terminal tail and the conserved CENP-A Targeting Domain (CATD) marked on the sequence. B Immunostaining of nucleus in the F. suspensa using the anti-CENH3 antibody. Arrows indicate CENH3 signals. Bar = 10 μm. C FISH assay using CENH3-ChIPed DNA as probes. The boxed insets show high-magnification image of the chromosome indicated by the arrow. Chromosomes are stained with DAPI. Bar = 10 μm. D This schematic overview illustrates the results of the CENH3 ChIP-seq assay in F. suspensa. The mapped reads density was plotted in 100-kb windows along the 14 chromosomes. The x-axis shows the positions on each chromosome. The y-axis represents ChIP/input ratio of the normalized density of uniquely mapped reads on each position, which was calculated based on the ratio of reads per million/100.

Genomic mapping of functional centromeres in F. suspensa

While utilizing centromere-specific repeats provides valuable resources, the precise determination of centromere positions is a concern, particularly in light of the extensive centromere repositioning phenomena observed across various species during evolution [20–24]. To accurately locate the centromeres within the F. suspensa genome, we developed a highly specific antibody against the single copy of the CENH3 gene (FsCENH3) identified in the genome (Fig. 3A). This antibody, crafted from a peptide that corresponds to the N-terminal segment of the FsCENH3 sequence, displayed the remarkable ability to precisely target all functional F. suspensa centromeres (Fig. 3B). We next harnessed chromatin immunoprecipitation sequencing (ChIP-seq) with the purpose-designed anti-CENH3 antibody for F. suspensa. The application of fluorescence in situ hybridization (FISH) with ChIPed DNA as the probe revealed distinct, strong signals at the centromeres, suggesting that we successfully captured the majority of genuine centromeric DNA in our samples (Fig. 3C). The DNA fragments successfully immunoprecipitated were subsequently subjected to high-throughput sequencing on the NovaSeq platform. This sequencing data was analysed alongside control DNA samples, carefully extracted from chromatin preparations before commencing the ChIP procedure. Our sequencing analysis generated an average of 16 million reads, achieving a coverage of 6× (Table S15, see online supplementary material). After stringent filtering, the refined reads were accurately aligned to the Fsus-CHAU genome. This unveiled a notable concentration of CENH3 peaks within the centromeric domains spanning all 14 chromosomes of F. suspensa (Fig. 3D). The core region of CENH3 binding in F. suspensa centromeres spans a total of 12.9 Mb, ranging from 0.4 to 1.5 Mb in size, which accounts for 1.87% of the 688.79 Mb F. suspensa genome, with a mean centromere size of approximately 0.9 Mb (Table 2). We also employed the CentroMiner module within the quarTeT software to identify centromere positions [25]. The results showed that quarTeT accurately identified centromere locations, consistent with functional centromere positions identified by CENH3-ChIP-seq (Fig. S3, see online supplementary material). In summary, we have successfully identified and characterized the functional centromeres in the F. suspensa genome.

Table 2 Locations of functional CENH3-binding sites in Forsythia suspensa.

Chr	Chr size (Mb)	Cen location (Mb)	Cen size (Mb)	CentFs arrays (Mb)	Chr	Chr size (Mb)	Cen location (Mb)	Cen size (Mb)	CentFs arrays (Mb)	
1	47	35.9–37.3	1.4	1.3	8	43	11.2–12.0	0.8	1.0	
2	51	16.7–17.5	0.8	3.0	9	54	24.4–25.4	1.0	3.7	
3	40	30.1–30.5	0.4	4.9	10	56	31.6–32.6	1.0	3.5	
4	64	19.0–20.5	1.5	3.2	11	62	41.5–42.3	0.8	1.0	
5	40	19.4–20.0	0.6	1.5	12	69	45.8–47.2	1.4	4.0	
6	46	19.8–21.3	1.5	1.5	13	53	19.9–20.7	0.8	2.8	
7	45	21.1–21.6	0.5	4.0	14	50	26.7–27.1	0.4	4.0	
		Cen, centromere; Chr, chromosome; CentFs, F. suspensa centromeric satellites.	

The centromeres contain rich and diverse satellite sequences in F. suspensa

To unravel the sequence composition of F. suspensa centromeres, we embarked on a comprehensive quest to characterize centromere-specific repetitive elements within the F. suspensa genome. Our approach began with utilizing RepeatExplorer to scrutinize and delineate repeat clusters. Among the clusters examined, 20 showed a CENH3-ChIP/input ratio surpassing 7, with their genomic representation exceeding 0.05% (Fig. S4A, see online supplementary material). Through the use of the Basic Local Alignment Search Tool (BLAST), we pinpointed four clusters that were specifically associated with the centromeres (Fig. S4B–E, see online supplementary material). All four clusters were identified as satellite repeats arranged in tandem, each with a distinct unit size: 356, 365, 530, and 732 base pairs, respectively (Fig. S4B–E, see online supplementary material). These were named CentFs356, CentFs365, CentFs530, and CentFs732 according to their respective lengths. We also utilized the de novo mode of the Tandem Repeat Annotation and Structural Hierarchy (TRASH) tool to investigate tandem repeats. This tool not only validated the existence of four distinct satellite populations as identified but also uncovered novel centromere-specific satellites. These satellites, particularly those with lengths around 360 bp and 730 bp, emerged as the predominant types (Fig. 4E and F). Interestingly, the primary satellites of centromeres varied across different chromosomes (Fig. 4B). For example, while CentFs54 was prevalent in centromere 1, CentFs727 was dominant in centromere 12 (Fig. 4B). Moreover, most centromeres exhibited a blend of satellites; for instance, centromere 4 was composed mainly of CentFs356 and CentFs365, whereas centromere 9 comprised primarily CentFs356, CentFs365, and CentFs733 (Fig. 4A). We selected four centromeric satellites and designed specific oligo probes. Subsequent FISH analyses unveiled the localization of all centromeric satellites to their respective centromeric regions (Fig. 4D; Fig. S5, see online supplementary material). Notably, variations in signal intensities were observed, suggesting differences in abundance among distinct centromeric regions (Fig. 4D). Additionally, dot plot analysis unveiled significant discrepancies in the genomic landscape of centromeres, underscoring the unique sequence and structural features of each centromere in F. suspensa (Fig. 4C). These findings highlight the intricate and diverse organization of centromeric satellites in F. suspensa, suggesting a dynamic evolutionary process with potentially multifaceted functions.

Figure 4 Characterization of the diverse centromeric satellites in Forsythia suspensa. A Characteristics of centromeres within F. suspensa genome assemblies. The enrichment of CENH3 [log2(ChIP/Input)] depicted across centromere 9. B A circos plot depicting tandem repeats identified by TRASH in de novo mode within the Fsus-CHAU genome assembly. C Dot plots comparing the centromeres using a search window of 100 bp. Forward- and reverse-strand similarity are indicated by different points. D FISH signals of CentFs356, CentFs365, and CentFs733 in F. suspensa. Bar = 10 μm. E A histogram illustrating the lengths (bp) of tandem repeats identified in the Fsus-CHAU genome assembly by TRASH. F Proportion of the genome occupied by different tandem repeats.

CENH3 nucleosomes exhibit a distinctive highly phased arrangement at specific sites along centromere satellites, a crucial aspect believed to contribute to proper centromere function [26–28]. To explore whether CENH3 nucleosomes in F. suspensa bind randomly to centromeric satellite sequences of large monomer size or target specific sites. Employing a methodology akin to centromere analyses in humans and plants [26, 29], we combined CENH3-ChIP reads, indicative of CENH3-bound nucleosomes, with input reads, representing bulk nucleosome fragments. These reads were subsequently aligned to consensus satellite sequences of trimer repeats (comprising three copies of a consensus monomer). The distribution of fragment midpoints was then utilized to determine the positioning of CENH3 or canonical nucleosomes. From the CENH3-ChIP reads, we observed five distinct binding peaks in the trimeric CentFs356 (Fig. S6A, see online supplementary material), and two prominent binding peaks in the trimeric CentFs365 (Fig. S6B, see online supplementary material), CentFs530 (Fig. S6C, see online supplementary material), and CentFs732 (Fig. S6D, see online supplementary material) consensus sequence. This suggests a phased arrangement of CENH3 nucleosomes on F. suspensa centromeric satellite sequences. Notably, the positions of input nucleosomes on the monomer mirrored those of the CENH3 nucleosomes (Fig. S6A–D, see online supplementary material), implying that a significant portion of repeats in CentFs356, CentFs365, CentFs530, and CentFs732 were enfolded around the CENH3 nucleosomes.

Asexual reproduction in F. suspensa may have impacted the dynamics of retrotransposon invasions, thereby driving the evolution of centromeres

We observed a notable disruption of satellite arrays attributed to retrotransposons (Fig. 5A). We meticulously annotated the full-length LTR-RTs within the genome of Fsus-CHAU. Our analysis revealed a total of 3471 full-length LTR-RTs, with 295 located specifically within the centromeric regions (Fig. 5F and G). Notably, we detected instances of fragmented and nested LTR insertions within these centromeres (Fig. 5A). A detailed examination, represented through dot plots, showcased substantial sequence divergence among centromeric full-length LTR-RTs (Fig. 5C), leading to their classification into seven distinct clades (Fig. 5B). Interestingly, these LTR-RTs clustered alongside those found outside the centromeres, indicating subsequent duplications post-integration within the centromeres (Fig. 5B). Furthermore, our investigation unveiled a higher enrichment level of CENH3 on the satellite arrays compared to the centromeric retrotransposons (Fig. 5D). This observation suggests that the repetitive nature or chromatin structure of satellite arrays potentially renders them more favorable binding sites for CENH3.

Figure 5 The LTR-RTs in the centromeres of Forsythia suspensa. A The distribution of full-length LTR-RTs, fragmented LTRs, and centromeric satellites within the extended 20 Mb region surrounding the centromere of Chromosome 7. B Phylogenetic tree of full-length LTR-RTs from the Fsus-CHAU genome, with branches differentiated by their locations within and outside the centromeres. C Dot plot of centromeric full-length LTR-RTs from the Fsus-CHAU genome using a 100-bp search window. D CENH3 enrichment level [log2(ChIP/Input)] around CentFs satellites, centromeric full-length LTR-RTs (n = 295) and non-centromeric full-length LTR-RTs (n = 3176). E Comparison of the insertion timing between full-length LTR-RTs located within centromeres (Cen_LTR-RTs) and those outside of centromeres (Non-cen_LTR-RTs). F Non-centromeric full-length LTR-RTs (Non-cen_LTR-RTs): the histogram displays the distribution of LTR identity percentages for non-cen_LTR-RTs, with a sample size of 3176 elements. The median LTR identity is 0.982, as indicated by the vertical red line. The x-axis represents the percentage of LTR identity, while the y-axis shows the count of LTR-RTs within each identity range. G Centromeric full-length LTR-RTs (Cen_LTR-RTs): the histogram shows the distribution of LTR identity percentages for centromeric LTR-RTs, with a sample size of 295 elements. The median LTR identity is 0.979, marked by the vertical red line. The x-axis represents the percentage of LTR identity, and the y-axis indicates the count of LTR-RTs within each identity range.

In assessing the insertion time of full-length LTRs within centromeric regions, we discovered that LTRs within the centromeres are older than those present across the entire genome (Fig. 5E). LTR identity can indicate the age of centromeric retrotransposons because when a retrotransposon is first inserted, its LTR sequences are identical. Over time, mutations accumulate independently in each LTR, leading to decreased sequence identity. Thus, lower LTR identity suggests a longer time since insertion, indicating an older age [30]. We analysed LTR identity between centromeric and non-centromeric regions. On average, LTR identities in non-centromeric regions were notably higher than those within centromeres, indicating the comparatively older age of centromeric retrotransposons (Fig. 5F and G). This finding contrasts with reports from other species, where very young CRM retrotransposons were observed in centromeres [31–34]. Recent studies have suggested a decrease in transposable element loads under asexual reproduction [35]. Given that F. suspensa predominantly reproduces asexually, primarily through methods like vegetative propagation rather than sexual reproduction, this raises intriguing possibilities that asexual reproduction could influence the dynamics of retrotransposon invasions, which in turn may have shaped the evolution of centromeres.

Discussion

The F. suspensa genome has long been a subject of interest, with recent advances in research shedding light on its complexities and challenges. F. suspensa faces obstacles arising from incomplete assembly in repetitive regions, hindering a comprehensive understanding of its genomic landscape. In response to these challenges, we undertook an extensive genome sequencing initiative, employing state-of-the-art technologies such as ONT sequencing, PacBio HiFi, and Hi-C. The resulting T2T assembly, denoted as Fsus-CHAU, represents a significant leap forward, surpassing previous genomes in continuity and completeness. Fsus-CHAU consists of 14 chromosomes with a total cumulative length of 688.79 Mb and an N50 length of 48.48 Mb. Similar to the utilization of reference genomes in other plant communities, Fsus-CHAU serves as a valuable resource for in-depth exploration of F. suspensa’s genomic intricacies.

Centromeres play a crucial role in ensuring accurate chromosome segregation and genetic material distribution during cell division. Unlike sequences found on chromosome arms, centromeres are distinguished by a dense concentration of repetitive elements specific to the centromeric region. Researchers frequently rely on these centromere-specific repetitive elements to pinpoint functional centromeres in T2T genomes. However, this approach encounters a significant challenge: centromeric satellite arrays are often larger than the actual functional centromeric regions. CENH3 immunolocalization provides a direct method to pinpoint functional centromeres. This methodological robustness is particularly valuable in studies involving species prone to centromere dynamics and genomic rearrangements. A key milestone in our study was the first-time identification of the CENH3 within the Forsythia genus. Furthermore, our study advanced beyond gene identification by taking a proactive step in characterizing functional centromeres in F. suspensa. This involved the development of a highly specific CENH3 antibody. The application of this antibody allowed us to precisely target and identify functional centromeric regions in F. suspensa. We found the average size of the core region of CENH3 binding in F. suspensa centromeres is 0.9 Mb, markedly smaller than the size of the F. suspensa centromeric satellite arrays (Table 2). These observations align with recent analyses conducted in soybean and Arabidopsis [17, 19]. This common pattern implies that the proportional relationship between the size of CENH3-enriched regions and the broader satellite arrays could serve as a shared organizational characteristic in centromeres across various plant species.

In our study, we investigated the centromere structure of F. suspensa and found that its centromeres are characterized by a high density of satellite DNA arrays, which are tandemly repeated sequences specific to the centromeric region. A small portion of LTR-RTs interspersed within these satellite arrays potentially influence centromere function and stability. This centromere structure is similar to that observed in grape [36, 37], soybean [19], maize [38], and Arabidopsis [39], which also have satellite-rich centromeres. In contrast, species such as wheat [32], oats [40], and rye [41] have centromeres that are rich in retrotransposons, lacking extensive satellite DNA arrays and instead being dominated by retrotransposon sequences. The differences between satellite-rich and retrotransposon-rich centromeres highlight the diversity of centromere evolution and its implications for genome dynamics. Satellite-rich centromeres, despite their disruptions by retrotransposons, maintain a high degree of sequence specificity and structural integrity essential for proper chromosome segregation. The interplay between satellite DNA and retrotransposons in these centromeres suggests a balance between maintaining centromere stability and allowing for evolutionary flexibility. On the other hand, retrotransposon-rich centromeres represent a more fluid and dynamic centromere architecture. The dominance of retrotransposons can lead to rapid centromere evolution and adaptation, potentially offering advantages in rapidly changing environments or under selective pressures. The identified satellites in F. suspensa exhibit a notable departure from the typical monomeric lengths associated with well-established centromeric repeats. Established repeats often consist of monomers ranging from 150 to 180 base pairs, exemplified by well-known sequences such as pAL1 in Arabidopsis [39] and CentC in maize [38]. Such monomeric lengths are crucial because they can accommodate a single CENH3 nucleosome, which in turn aids in the assembly of CENH3 nucleosome arrays with a specific arrangement throughout the chromosome, essential for the establishment and stability of centromeres. Contrastingly, the newly identified centromeric satellites in F. suspensa showcase a wide range of monomeric lengths, spanning from 300 base pairs to 700 base pairs. This variation, often characterized as ‘odd’ repeats, has been observed to be limited to specific centromeres and not universally present across all centromeres. The unconventional lengths of these repeats suggest a dynamic process of amplification and adaptation. This dynamic nature raises intriguing questions about the functional implications of such satellite structures in centromere biology.

Based on our findings, the age distribution and identity of LTRs within centromeric regions present intriguing insights into the dynamics of retrotransposon invasions and their impact on centromere evolution in F. suspensa. Contrary to observations in other species where young CRM retrotransposons dominate centromeric regions, our study reveals a striking pattern: LTRs within the centromeres of F. suspensa are notably older than those across the entire genome (Fig. 5E–G). This departure from the norm prompts a reevaluation of the interplay between asexual reproduction and retrotransposon dynamics. The prevalence of asexual reproduction in F. suspensa, primarily through mechanisms such as vegetative propagation, suggests a potential link between reproductive strategy and retrotransposon dynamics. Recent studies have hinted at a reduction in transposable element loads under asexual reproduction conditions [35]. Our findings add weight to this hypothesis by indicating that asexual reproduction might influence the dynamics of retrotransposon invasions, thereby shaping the evolutionary trajectory of centromeres. This departure from the expected pattern underscores the complexity of centromere evolution and highlights the need for a nuanced understanding of the interplay between genomic dynamics and reproductive strategies. Furthermore, our study opens up avenues for exploring the mechanisms underlying retrotransposon regulation and their role in shaping centromere architecture across different species. Future research could delve deeper into the molecular mechanisms mediating retrotransposon dynamics under varying reproductive strategies, shedding light on the broader evolutionary implications for genome organization and stability in diverse taxa.

Materials and methods

Plant materials, DNA library construction, and sequencing

The wild F. suspensa was cultivated within a greenhouse environment under controlled conditions for 30 days, maintaining a 16-hour light and 8-hour dark cycle at a temperature of 28°C. For Oxford Nanopore Ultra-long sequencing, the libraries were constructed according to standard protocols and sequencing was conducted using a PromethION sequencer by Oxford Nanopore Technologies (Oxford, Oxfordshire, UK). For PacBio HiFi sequencing, libraries were prepared using the Pacific Biosciences SMRT bell express template prep kit 2.0 following the standard protocol. Subsequently, a SMRT cell sequencing library was created, containing approximately 15 kb cut fragments, and sequenced using the PacBio sequel II sequencing platform with 30-hour runs (Pacific Biosciences, Menlo Park, CA, USA). For Hi-C sequencing, we utilized high-quality DNA extracted from the youthful leaves of F. suspensa species. Chromatin was appropriately fixed using formaldehyde, and the in situ Hi-C chromosome conformation capture procedure was executed following the DNase-based protocol originally outlined by Ramani [42]. Subsequently, sequencing of the Hi-C libraries was conducted using the Illumina NovaSeq platform in paired-end 150-bp sequence reads (Illumina, San Diego, CA, USA). For Illumina RNA sequencing, the leaf samples were used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (#E7530L, NEB, USA) following the manufacturer’s recommendations, then the libraries were sequenced on an Illumina platform and 150 bp paired-end reads were generated, all of which were conducted at Wuhan Benagen Technology Co., Ltd in Wuhan, China.

Genome assembly

The genome assembly employed various strategies based on different algorithms, resulting in four primary sets of contig genomes. Consensus reads were filtered using the CCS software with default parameters. High-quality HiFi reads were utilized for assembly through Hifiasm with default parameters, resulting in a draft genome [43]. ONT ultralong sequencing reads were filtered, and only those of high quality were employed for assembly using NextDenovo [44] with default parameters.

Following initial assembly, Racon [45] and Pilon [46] were applied for three rounds of correction and polishing with ONT and Illumina PE reads, yielding the final contigs. Hi-C data were processed using fastp [47] to clean the data, while HiCUP extracted valid interaction pairs to facilitate chromosome-level assembly. Subsequently, ALLHiC [48] and Juicebox [49] clustered, ordered, and oriented the contigs. The Hifiasm-generated assembly served as the backbone genome, with the ONT genome filling gaps in the backbone. The genome underwent further correction with aligned HiFi reads using Winnowmap. Finally, a genomic interaction heatmap for the T2T genome was generated using HiCExplorer [50].

Genes and transposable elements annotation

For transposable elements annotation, the de novo approach was employed using the RepeatModeler software [51] to predict model sequences from the genomic sequence. Simultaneously, the LTR_FINDER software [52] and LTRharvest [53] were used to predict LTR sequences. The LTR_retriever software [54] was then applied to remove redundancy from the results obtained by the two aforementioned methods, resulting in a non-redundant set of LTR sequences. The de novo repeat sequence library was created by merging the results of these two de novo sequence prediction methods. The teclass software [55] was utilized to redefine the unknown content within this library. Subsequently, the RepBase library and the de novo library were merged, and the RepeatMasker software was employed to align and predict repetitive sequences, yielding the de novo + repbase results. Finally, the results of all repetitive predictions were consolidated, and redundant entries were eliminated to yield the final dispersed repetitive sequence set, referred to as Combined TEs.

Gene annotation encompassed a comprehensive approach, integrating ab initio predictions, homology-based methods, and RNA-seq data-supported strategies. Two ab initio gene prediction tools, Augustus [56] and GlimmerHMM [57], were employed, with BUSCO [58] aiding in the acquisition of training sets for these predictors. Homology-based gene prediction involved the selection of reference species, including Fraxinus pennsylvanica, Olea europaea, Jasminum sambac, and Osmanthus fragrans with Exonerate used for the prediction. RNA-seq reads underwent filtration using fastp and were aligned to the assembled genome through hisat2 [59]. Afterward, gene models were predicted using StringTie [60] and TransDecoder. This integrated approach aimed to enhance the accuracy and completeness of gene annotation by leveraging the strengths of multiple prediction methods and supporting evidence from RNA-seq data.

CENH3-ChIP-seq library construction and data analysis

CENH3-ChIP-seq was conducted as previously described [40]. Approximately 5 g of young leaves from F. suspensa were utilized for the ChIP experiment. The libraries underwent sequencing on the Illumina NovaSeq platform to produce paired-end 150-bp sequence reads. The processing and alignment of CENH3-ChIP-Seq raw data were conducted as previously described [19].

Identifying and characterizing the centromeric repeats

A total of one million randomly selected sequence reads (150 bp) from the input control were analysed using the web-based GALAXY REPEATEXPLORER (https://repeatexplorer-elixir.cerit-sc.cz/galaxy/) with default parameters. This analysis aimed to scrutinize and delineate repeat clusters. The repeats were identified and classified into individual repeat clusters based on their sequence similarity. To estimate the genome proportion of each repeat cluster, we counted the number of reads in each cluster. This step provided us with the genomic representation of each repeat cluster.

Next, to identify the repeats associated with the CENH3-containing chromatin, we conducted a BLAST analysis. The CENH3 ChIP-seq and input reads were separately subjected to BLASTn analysis against the repeat clusters using an E-value threshold of 1e–8. This process enabled us to determine which repeat sequences were enriched in the CENH3 ChIP-seq data compared to the input control. The CENH3-ChIP/input ratio was calculated by comparing the number of aligned reads from the CENH3 ChIP-seq data to the number of aligned reads from the input data for each repeat cluster. This ratio represents the relative enrichment of each repeat cluster in the CENH3 ChIP-seq data. A higher ratio indicates a greater association of that repeat cluster with CENH3-containing chromatin.

LTR retrotransposon insertion time

Estimating the insertion time of LTR retrotransposons involves analysing the divergence between their two LTR sequences, which accumulate mutations after integration into the host genome. This process utilizes sequence alignment tools to compare the LTR sequences and calculate their differences or percent divergence. By knowing the mutation rate of LTR sequences, typically estimated from known rates in related species, the insertion time can be approximated using the formula T = K/ (2 * r), where K is the number of differences and r is the mutation rate per site per year [30].

Supplementary Material

Web_Material_uhae185

Acknowledgments

This work was funded by the National Natural Science Foundation of China (32360307 and 32300491).

Author contributions

Yang Liu., Yong Li., F.H., and J.C. designed the experiments and managed the project. J.C., C.Z., L.S., C.Y., R.W., X.S., F.H., Y.L., and Y.L. performed the data analyses. Y.L. and J.C. wrote the manuscript. All authors have reviewed and approved the final version of the manuscript.

Data availability statement

The genome sequence data, the genome assembly, and the associated annotation have been deposited at the National Genomics Data Center and can be accessed with the accession code PRJCA023056.

Conflict of interests

No conflict of interest was declared.

Supplementary data

Supplementary data is available at Horticulture Research online.
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