Gapless Genome Research Articles

Pervasive conservation of intron number and other genetic elements revealed by a chromosome-level genomic assembly of the hyper-polymorphic nematode Caenorhabditis brenneri.

With within-species genetic diversity estimates that span the gambit of that seen across the entirety of animals, the Caenorhabditis genus of nematodes holds unique potential to provide insights into how population size and reproductive strategies influence gene and genome organization and evolution. Our study focuses on Caenorhabditis brenneri, currently known as one of the most genetically diverse nematodes within its genus and metazoan phyla. Here, we present a high-quality gapless genome assembly and annotation for C. brenneri, revealing a common nematode chromosome arrangement characterized by gene-dense central regions and repeat rich peripheral parts. Comparison of C. brenneri with other nematodes from the 'Elegans' group revealed conserved macrosynteny but a lack of microsynteny, characterized by frequent rearrangements and low correlation iof orthogroup sizes, indicative of high rates of gene turnover. We also assessed genome organization within corresponding syntenic blocks in selfing and outcrossing species, affirming that selfing species predominantly experience loss of both genes and intergenic DNA. Comparison of gene structures revealed strikingly small number of shared introns across species, yet consistent distributions of intron number and length, regardless of population size or reproductive mode, suggesting that their evolutionary dynamics are primarily reflective of functional constraints. Our study provides valuable insights into genome evolution and expands the nematode genome resources with the highly genetically diverse C. brenneri, facilitating research into various aspects of nematode biology and evolutionary processes.

The gapless genome assembly and multi-omics analyses unveil a pivotal regulatory mechanism of oil biosynthesis in the olive tree.

Olive is a valuable oil-bearing tree with fruits containing high levels of fatty acids. Oil production is a multifaceted process involving intricate interactions between fatty acid biosynthesis and other metabolic pathways that are affected by genetics and the developmental stages of the fruit. However, a comprehensive understanding of the underlying regulatory mechanisms is still lacking. Here, we generated a gap-free telomere-to-telomere assembly for Olea europaea cv. 'Leccino', representing an olive genome with the highest contiguity and completeness to date. The combination of time-course metabolomics and transcriptomics datasets revealed a negative correlation between fatty acid and flavonoid biosynthesis in the initial phase of olive fruit development, which was subject to an opposing regulatory mechanism mediated by the hub transcription factor MYC2. Multifaceted molecular assays demonstrated that MYC2 is a repressor of fatty acid biosynthesis by downregulating the expression of BCCP2 (biotin carboxylase carrier protein 2), while it acts as an activator of FLS (flavonol synthase), leading to an increase in flavonoid synthesis. Furthermore, the expression of MYC2 is regulated by fluctuations of methyl jasmonate content during olive fruit development. Our study completes a high-quality gapless genome of an olive cultivar, and provides new insight into the regulatory mechanisms underlying the biosynthesis of fatty acids and flavonoids in its fruit.

Haplotype-resolved gapless genome and chromosome segment substitution lines facilitate gene identification in wild rice

The abundant genetic variation harbored by wild rice (Oryza rufipogon) has provided a reservoir of useful genes for rice breeding. However, the genome of wild rice has not yet been comprehensively assessed. Here, we report the haplotype-resolved gapless genome assembly and annotation of wild rice Y476. In addition, we develop two sets of chromosome segment substitution lines (CSSLs) using Y476 as the donor parent and cultivated rice as the recurrent parents. By analyzing the gapless reference genome and CSSL population, we identify 254 QTLs associated with agronomic traits, biotic and abiotic stresses. We clone a receptor-like kinase gene associated with rice blast resistance and confirm its wild rice allele improves rice blast resistance. Collectively, our study provides a haplotype-resolved gapless reference genome and demonstrates a highly efficient platform for gene identification from wild rice.

A telomere-to-telomere gap-free reference genome assembly of avocado provides useful resources for identifying genes related to fatty acid biosynthesis and disease resistance.

Avocado (Persea americana Mill.) is an economically valuable plant because of the high fatty acid content and unique flavor of its fruits. Its fatty acid content, especially the relatively high unsaturated fatty acid content, provides significant health benefits. We herein present a telomere-to-telomere gapless genome assembly (841.6Mb) of West Indian avocado. The genome contains 40 629 predicted protein-coding genes. Repeat sequences account for 57.9% of the genome. Notably, all telomeres, centromeres, and a nucleolar organizing region are included in this genome. Fragments from these three regions were observed via fluorescence in situ hybridization. We identified 376 potential disease resistance-related nucleotide-binding leucine-rich repeat genes. These genes, which are typically clustered on chromosomes, may be derived from gene duplication events. Five NLR genes (Pa11g0262, Pa02g4855, Pa07g3139, Pa07g0383, and Pa02g3196) were highly expressed in leaves, stems, and fruits, indicating they may be involved in avocado disease responses in multiple tissues. We also identified 128 genes associated with fatty acid biosynthesis and analyzed their expression patterns in leaves, stems, and fruits. Pa02g0113, which encodes one of 11 stearoyl-acyl carrier protein desaturases mediating C18 unsaturated fatty acid synthesis, was more highly expressed in the leaves than in the stems and fruits. These findings provide valuable insights that enhance our understanding of fatty acid biosynthesis in avocado.

First telomere-to-telomere gapless assembly of the rice blast fungus Pyricularia oryzae

Rice blast caused by Pyricularia oryzae (syn., Magnaporthe oryzae) was one of the most destructive diseases of rice throughout the world. Genome assembly was fundamental to genetic variation identification and critically impacted the understanding of its ability to overcome host resistance. Here, we report a gapless genome assembly of rice blast fungus P. oryzae strain P131 using PacBio, Illumina and high throughput chromatin conformation capture (Hi-C) sequencing data. This assembly contained seven complete chromosomes (43,237,743 bp) and a circular mitochondrial genome (34,866 bp). Approximately 14.31% of this assembly carried repeat sequences, significantly greater than its previous assembled version. This assembly had a 99.9% complement in BUSCO evaluation. A total of 14,982 genes protein-coding genes were predicted. In summary, we assembled the first telomere-to-telomere gapless genome of P. oryzae, which would be a valuable genome resource for future research on the genome evolution and host adaptation.

Genome Comparisons between Botrytis fabae and the Closely Related Gray Mold Fungus Botrytis cinerea Reveal Possible Explanations for Their Contrasting Host Ranges.

While Botrytis cinerea causes gray mold on many plants, its close relative, Botrytis fabae, is host-specifically infecting predominantly faba bean plants. To explore the basis for its narrow host range, a gapless genome sequence of B. fabae strain G12 (BfabG12) was generated. The BfabG12 genome encompasses 45.0 Mb, with 16 chromosomal telomere-to-telomere contigs that show high synteny and sequence similarity to the corresponding B. cinerea B05.10 (BcB0510) chromosomes. Compared to BcB0510, it is 6% larger, due to many AT-rich regions containing remnants of transposable elements, but encodes fewer genes (11,420 vs. 11,707), due to losses of chromosomal segments with up to 20 genes. The coding capacity of BfabG12 is further reduced by nearly 400 genes that had been inactivated by mutations leading to truncations compared to their BcB0510 orthologues. Several species-specific gene clusters for secondary metabolite biosynthesis with stage-specific expression were identified. Comparison of the proteins secreted during infection revealed high similarities, including 17 phytotoxic proteins that were detected in both species. Our data indicate that evolution of the host-specific B. fabae occurred from an ancestral pathogen with wide host range similar to B. cinerea and was accompanied by losses and degeneration of genes, thereby reducing its pathogenic flexibility.

Characterization of O-methyltransferases in the biosynthesis of phenylphenalenone phytoalexins based on the telomere-to-telomere gapless genome of Musella lasiocarpa.

Phenylphenalenones (PhPNs), phytoalexins in wild bananas (Musaceae), are known to act against various pathogens. However, the abundance of PhPNs in many Musaceae plants of economic importance is low. Knowledge of the biosynthesis of PhPNs and the application of biosynthetic approaches to improve their yield is vital for fighting banana diseases. However, the processes of PhPN biosynthesis, especially those involved in methylation modification, remain unclear. Musella lasiocarpa is a herbaceous plant belonging to Musaceae, and due to the abundant PhPNs, their biosynthesis in M. lasiocarpa has been the subject of much attention. In this study, we assembled a telomere-to-telomere gapless genome of M. lasiocarpa as the reference, and further integrated transcriptomic and metabolomic data to mine the candidate genes involved in PhPN biosynthesis. To elucidate the diversity of PhPNs in M. lasiocarpa, three screened O-methyltransferases (Ml01G0494, Ml04G2958, and Ml08G0855) by phylogenetic and expressional clues were subjected to in vitro enzymatic assays. The results show that the three were all novel O-methyltransferases involved in the biosynthesis of PhPN phytoalexins, among which Ml08G0855 was proved to function as a multifunctional enzyme targeting multiple hydroxyl groups in PhPN structure. Moreover, we tested the antifungal activity of PhPNs against Fusarium oxysporum and found that the methylated modification of PhPNs enhanced their antifungal activity. These findings provide valuable genetic resources in banana breeding and lay a foundation for improving disease resistance through molecular breeding.

Reinstatement of the fission yeast species Schizosaccharomyces versatilis Wickerham et Duprat, a sibling species of Schizosaccharomyces japonicus.

Schizosaccharomyces japonicus Yukawa et Maki (1931) and Schizosaccharomyces versatilis Wickerham et Duprat (1945) have been treated as varieties of S. japonicus or as conspecific, based on various approaches including mating trials and nDNA/nDNA optical reassociation studies. However, the type strains of S. japonicus and S. versatilis differ by five substitutions (99.15% identity) and one 1-bp indel in the sequences of the D1/D2 domain of the 26S rRNA gene, and 23 substitutions (96.3% identity) and 31-bp indels in the sequences of internal transcribed spacer (ITS) of rRNA, suggesting that they may not be conspecific. To reassess their taxonomic status, we conducted mating trials and whole-genome analyses. Mating trials using the type strains showed a strong but incomplete prezygotic sterility barrier, yielding interspecies mating products at two orders of magnitude lower efficiency than intraspecies matings. These mating products, which were exclusively allodiploid hybrids, were unable to undergo the haplontic life cycle of the parents. We generated chromosome-level gap-less genome assemblies for both type strains. Whole genome sequences yielded an average nucleotide identity (ANI) of 86.4%, indicating clear separation of S. japonicus and S. versatilis. Based on these findings, we propose the reinstatement of S. versatilis as a distinct species (holotype strain: CBS 103T and ex-types: NRRL Y-1026, NBRC 1607, ATCC 9987, PYCC 7100; Mycobank no.: 847838).

The Complete and Gapless Genome Sequence of the Sugarbeet Pathogen Cercospora beticola

Cercospora beticola, causal agent of Cercospora leaf spot of sugarbeet ( Beta vulgaris subsp. vulgaris), is the most economically damaging foliar pathogen inhibiting sugarbeet production. Despite the global importance of C. beticola, significant knowledge gaps exist pertaining to pathogen virulence and adaptation to management practices that include the use of resistant host varieties and the application of different fungicide chemistries. The lack of a high-quality reference genome for C. beticola has hindered research progression addressing these critical knowledge gaps. Here, we report the complete and gapless genome assembly and annotation of the reference C. beticola isolate Cb09-40. We identified misassembled regions in the previous reference genome and annotated previously missing protein-coding genes. This work establishes a high-quality reference genome that can serve as the foundation for future comparative genomic, population genetic, and evolutionary studies into the virulence and evolution of this important plant pathogen. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Gapless Genome Assembly of ZH8015 and Preliminary Multi-Omics Analysis to Investigate ZH8015’s Responses Against Brown Planthopper Infestation

Accurate genomic information is essential for advancing genetic breeding research in specific rice varieties. This study presented a gapless genome assembly of the indica rice cultivar Zhonghui 8015 (ZH8015) using PacBio HiFi, Hi-C, and ONT (Oxford Nanopore Technologies) ultra-long sequencing technologies, annotating 43 037 gene structures. Subsequently, utilizing this genome along with transcriptomic and metabolomic techniques, we explored ZH8015’s response to brown planthopper (BPH) infestation. Continuous transcriptomic sampling indicated significant changes in gene expression levels around 48 h after BPH feeding. Enrichment analysis revealed particularly significant alterations in genes related to reactive oxygen species scavenging and cell wall formation. Metabolomic results demonstrated marked increases in levels of several monosaccharides, which are components of the cell wall and dramatic changes in flavonoid contents. Omics association analysis identified differentially expressed genes associated with key metabolites, shedding light on ZH8015’s response to BPH infestation. In summary, this study constructed a reliable genome sequence resource for ZH8015, and the preliminary multi-omics results will guide future insect-resistant breeding research.

Telomere-to-telomere gapless genome assembly of the Chinese sea bass (Lateolabrax maculatus)

Chinese sea bass (Lateolabrax maculatus) is a highly sought-after commercial seafood species in Asian regions due to its excellent nutritional value. With the rapid advancement of bioinformatics, higher standards for genome analysis compared to previously published reference genomes are now necessary. This study presents a gapless assembly of the Chinese sea bass genome, which has a length of 632.75 Mb. The sequences were assembled onto 24 chromosomes with a coverage of over 99% (626.61 Mb), and telomeres were detected on 34 chromosome ends. Analysis using Merqury indicated a high level of accuracy, with an average consensus quality value of 54.25. The ONT ultralong and PacBio HiFi data were aligned with the assembly using minimap2, resulting in a mapping rate of 99.9%. The study also identified repeating elements in 20.90% (132.25 Mb) of the genome and inferred 22,014 protein-coding genes. These results establish meaningful groundwork for exploring the evolution of the Chinese sea bass genome and advancing molecular breeding techniques.

Mechanisms of Rapid Karyotype Evolution in Mammals.

Chromosome reshuffling events are often a foundational mechanism by which speciation can occur, giving rise to highly derivative karyotypes even amongst closely related species. Yet, the features that distinguish lineages prone to such rapid chromosome evolution from those that maintain stable karyotypes across evolutionary time are still to be defined. In this review, we summarize lineages prone to rapid karyotypic evolution in the context of Simpson's rates of evolution-tachytelic, horotelic, and bradytelic-and outline the mechanisms proposed to contribute to chromosome rearrangements, their fixation, and their potential impact on speciation events. Furthermore, we discuss relevant genomic features that underpin chromosome variation, including patterns of fusions/fissions, centromere positioning, and epigenetic marks such as DNA methylation. Finally, in the era of telomere-to-telomere genomics, we discuss the value of gapless genome resources to the future of research focused on the plasticity of highly rearranged karyotypes.

Insights into the evolution and spatial chromosome architecture of jujube from an updated gapless genome assembly

Insights into the evolution and spatial chromosome architecture of jujube from an updated gapless genome assembly

Telomere-to-telomere carrot (Daucus carota) genome assembly reveals carotenoid characteristics.

Carrot (Daucus carota) is an Apiaceae plant with multi-colored fleshy roots that provides a model system for carotenoid research. In this study, we assembled a 430.40Mb high-quality gapless genome to the telomere-to-telomere (T2T) level of "Kurodagosun" carrot. In total, 36 268 genes were identified and 34 961 of them were functionally annotated. The proportion of repeat sequences in the genome was 55.3%, mainly long terminal repeats. Depending on the coverage of the repeats, 14 telomeres and 9 centromeric regions on the chromosomes were predicted. A phylogenetic analysis showed that carrots evolved early in the family Apiaceae. Based on the T2T genome, we reconstructed the carotenoid metabolic pathway and identified the structural genes that regulate carotenoid biosynthesis. Among the 65 genes that were screened, 9 were newly identified. Additionally, some gene sequences overlapped with transposons, suggesting replication and functional differentiation of carotenoid-related genes during carrot evolution. Given that some gene copies were barely expressed during development, they might be functionally redundant. Comparison of 24 cytochrome P450 genes associated with carotenoid biosynthesis revealed the tandem or proximal duplication resulting in expansion of CYP gene family. These results provided molecular information for carrot carotenoid accumulation and contributed to a new genetic resource.

Gapless genome assembly of Fusarium verticillioides, a filamentous fungus threatening plant and human health

Fusarium verticillioides is a filamentous fungus that causes plant diseases and harms human health through cancer-inducing mycotoxin and life-threatening Fusariosis. Given its threat to agriculture and public health, genome assembly of this fungus is critical to our understanding of its pathobiology and developing antifungal drugs. Here, we report a gap-free genome assembly of F. verticillioides using PacBio HiFi data and high-throughput chromosome capture (Hi-C) sequencing data. The assembled 42.0 Mb sequence contains eleven gapless chromosomes capturing all centromeres and 19 of all 22 telomeres. This assembly represents a significant improvement over previous version on contiguity (contig N50: 4.3 Mb), completeness (BUSCO score: 99.0%) and correctness (QV: 88.8). A total of 15,230 protein-coding genes were predicted, 6.2% of which are newly annotated genes. In addition, we identified three-dimension chromatin structures such as TADs-like structures and chromatin loops based on Hi-C data of ultra-high coverage. This gap-free genome of F. verticillioides is an excellent resource for further panoramic understanding mechanisms of fungal genome evolution, mycotoxin production and pathogenesis on plant and human host.

Overexpression of Pericentromeric HSAT2 DNA Increases Expression of EMT Markers in Human Epithelial Cancer Cell Lines.

Pericentromeric tandemly repeated DNA of human satellites 1, 2, and 3 (HS1, HS2, and HS3) is actively transcribed in some cells. However, the functionality of the transcription remains obscure. Studies in this area have been hampered by the absence of a gapless genome assembly. The aim of our study was to map a transcript that we have previously described as HS2/HS3 on chromosomes using a newly published gapless genome assembly T2T-CHM13, and create a plasmid overexpressing the transcript to assess the influence of HS2/HS3 transcription on cancer cells. We report here that the sequence of the transcript is tandemly repeated on nine chromosomes (1, 2, 7, 9, 10, 16, 17, 22, and Y). A detailed analysis of its genomic localization and annotation in the T2T-CHM13 assembly revealed that the sequence belonged to HSAT2 (HS2) but not to the HS3 family of tandemly repeated DNA. The transcript was found on both strands of HSAT2 arrays. The overexpression of the HSAT2 transcript increased the transcription of the genes encoding the proteins involved in the epithelial-to-mesenchymal transition, EMT (SNAI1, ZEB1, and SNAI2), and the genes that mark cancer-associated fibroblasts (VIM, COL1A1, COL11A1, and ACTA2) in cancer cell lines A549 and HeLa. Co-transfection of the overexpression plasmid and antisense nucleotides eliminated the transcription of EMT genes observed after HSAT2 overexpression. Antisense oligonucleotides also decreased transcription of the EMT genes induced by tumor growth factor beta 1 (TGFβ1). Thus, our study suggests HSAT2 lncRNA transcribed from the pericentromeric tandemly repeated DNA is involved in EMT regulation in cancer cells.

Gapless Genome Assembly of Puccinia triticina Provides Insights into Chromosome Evolution in Pucciniales.

Chromosome evolution drives species evolution, speciation, and adaptive radiation. Accurate genome assembly is crucial to understanding chromosome evolution of species, such as dikaryotic fungi. Rust fungi (Pucciniales) in dikaryons represent the largest group of plant pathogens, but the evolutionary process of adaptive radiation in Pucciniales remains poorly understood. Here, we report a gapless genome for the wheat leaf rust fungus Puccinia triticina determined using PacBio high-fidelity (HiFi) sequencing. This gapless assembly contains two sets of chromosomes, showing that one contig represents one chromosome. Comparisons of homologous chromosomes between the phased haplotypes revealed that highly frequent small-scale sequence divergence shapes haplotypic variation. Genome analyses of Puccinia triticina along with other rusts revealed that recent transposable element bursts and extensive segmental gene duplications synergistically highlight the evolution of chromosome structures. Comparative analysis of chromosomes indicated that frequent chromosomal rearrangements may act as a major contributor to rapid radiation of Pucciniales. This study presents the first gapless, phased assembly for a dikaryotic rust fungus and provides insights into adaptive evolution and species radiation in Pucciniales. IMPORTANCE Rust fungi (Pucciniales) are the largest group of plant pathogens. Adaptive radiation is a predominant feature in Pucciniales evolution. Chromosome evolution plays an important role in adaptive evolution. Accurate chromosome-scale assembly is required to understand the role of chromosome evolution in Pucciniales. We took advantage of HiFi sequencing to construct a gapless, phased genome for Puccinia triticina. Further analyses revealed that the evolution of chromosome structures in rust lineage is shaped by the combination of transposable element bursts and segmental gene duplications. Chromosome comparisons of Puccinia triticina and other rusts suggested that frequent chromosomal arrangements may make remarkable contributions to high species diversity of rust fungi. Our results present the first gapless genome for Pucciniales and shed light on the feature of chromosome evolution in Pucciniales.

Chromosomal genome sequence assembly and mating-type (MAT) locus characterization of the leprose asexual lichenized fungus Lepraria neglecta (Nyl.) Erichsen

AbstractComplete chromosomal-level assemblies of fungal genomes are rare. The intimate ecological symbioses and complex reproduction strategies utilized by fungi make highly contiguous, gapless genome assemblies particularly difficult. Here, we use long-read sequencing on the Oxford Nanopore Technology MinION platform to sequence and assemble the genome of Lepraria neglecta (Ascomycota, Lecanorales). In addition to eight contigs ascribable to chromosomes, six of which are assembled telomere-to-telomere, we discovered the presence of a complete MAT locus with two conserved MAT1-2 genes and a putative MAT1-1 pseudogene. The full genome assembly of a widespread, common species presents an opportunity for new insights into lichen reproduction while the presence of the mating-type locus in the genome of an asexual lichen raises fundamental questions about reproductive biology in fungi generally.

Gapless genome assembly of East Asian finless porpoise

In recent years, conservation efforts have increased for rare and endangered aquatic wildlife, especially cetaceans. However, the East Asian finless porpoise (Neophocaena asiaeorientalis sunameri), which has a wide distribution in China, has received far less attention and protection. As an endangered small cetacean, the lack of a chromosomal-level reference for the East Asian finless porpoise limits our understanding of its population genetics and conservation biology. To address this issue, we combined PacBio HiFi long reads and Hi-C sequencing data to generate a gapless genome of the East Asian finless porpoise that is approximately 2.5 Gb in size over its 21 autosomes and two sex chromosomes (X and Y). A total of 22,814 protein-coding genes were predicted where ~97.31% were functionally annotated. This high-quality genome assembly of East Asian finless porpoise will not only provide new resources for the comparative genomics of cetaceans and conservation biology of threatened species, but also lay a foundation for more speciation, ecology, and evolutionary studies.Measurement(s)Neophocaena asiaeorientalis sunameri • Gapless genome assembly • sequence annotationTechnology Type(s)MGISEQ. 2000 • PacBio HiFi Sequencing • Hi-CSample Characteristic - OrganismNeophocaena asiaeorientalis sunameriSample Characteristic - EnvironmentseawaterSample Characteristic - LocationYellow Sea near Lianyungang City, Jiangsu Province, China

Gapless genome assembly of azalea and multi-omics investigation into divergence between two species with distinct flower color.

The genus Rhododendron (Ericaceae), with more than 1000 species highly diverse in flower color, is providing distinct ornamental values and a model system for flower color studies. Here, we investigated the divergence between two parental species with different flower color widely used for azalea breeding. Gapless genome assembly was generated for the yellow-flowered azalea, Rhododendron molle. Comparative genomics found recent proliferation of long terminal repeat retrotransposons (LTR-RTs), especially Gypsy, has resulted in a 125Mb (19%) genome size increase in species-specific regions, and a significant amount of dispersed gene duplicates (13402) and pseudogenes (17 437). Metabolomic assessment revealed that yellow flower coloration is attributed to the dynamic changes of carotenoids/flavonols biosynthesis and chlorophyll degradation. Time-ordered gene co-expression networks (TO-GCNs) and the comparison confirmed the metabolome and uncovered the specific gene regulatory changes underpinning the distinct flower pigmentation. B3 and ERF TFs were found dominating the gene regulation of carotenoids/flavonols characterized pigmentation in R. molle, while WRKY, ERF, WD40, C2H2, and NAC TFs collectively regulated the anthocyanins characterized pigmentation in the red-flowered R simsii. This study employed a multi-omics strategy in disentangling the complex divergence between two important azaleas and provided references for further functional genetics and molecular breeding.