The centromere is the chromosome region where microtubules attach during cell division. In contrast to monocentric chromosomes with one centromere, holocentric species usually distribute hundreds of centromere units along the entire chromatid. We assembled the chromosome-scale reference genome and analyzed the holocentromere and (epi)genome organization of the lilioid Chionographis japonica. Remarkably, each of its holocentric chromatids consists of only 7 to 11 evenly spaced megabase-sized centromere-specific histone H3-positive units. These units contain satellite arrays of 23 and 28 bp-long monomers capable of forming palindromic structures. Like monocentric species, C. japonica forms clustered centromeres in chromocenters at interphase. In addition, the large-scale eu- and heterochromatin arrangement differs between C. japonica and other known holocentric species. Finally, using polymer simulations, we model the formation of prometaphase line-like holocentromeres from interphase centromere clusters. Our findings broaden the knowledge about centromere diversity, showing that holocentricity is not restricted to species with numerous and small centromere units.

• MeSH
• Cell Division MeSH
• Centromere * genetics   MeSH
• Chromatids MeSH
• Heterochromatin genetics   MeSH
• Cell Cycle Proteins * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Heterochromatin MeSH
• Cell Cycle Proteins * MeSH

Tandem repeats are important parts of eukaryotic genomes being crucial e.g., for centromere and telomere function and chromatin modulation. In Lepidoptera, knowledge of tandem repeats is very limited despite the growing number of sequenced genomes. Here we introduce seven new satellite DNAs (satDNAs), which more than doubles the number of currently known lepidopteran satDNAs. The satDNAs were identified in genomes of three species of Crambidae moths, namely Ostrinia nubilalis, Cydalima perspectalis, and Diatraea postlineella, using graph-based computational pipeline RepeatExplorer. These repeats varied in their abundance and showed high variability within and between species, although some degree of conservation was noted. The satDNAs showed a scattered distribution, often on both autosomes and sex chromosomes, with the exception of both satellites in D. postlineella, in which the satDNAs were located at a single autosomal locus. Three satDNAs were abundant on the W chromosomes of O. nubilalis and C. perspectalis, thus contributing to their differentiation from the Z chromosomes. To provide background for the in situ localization of the satDNAs, we performed a detailed cytogenetic analysis of the karyotypes of all three species. This comparative analysis revealed differences in chromosome number, number and location of rDNA clusters, and molecular differentiation of sex chromosomes.

• Keywords
• Lepidoptera, W chromatin, holocentric chromosomes, repetitive DNAs, tandem repeat,
• Publication type
• Journal Article MeSH

Amplification of monomer sequences into long contiguous arrays is the main feature distinguishing satellite DNA from other tandem repeats, yet it is also the main obstacle in its investigation because these arrays are in principle difficult to assemble. Here we explore an alternative, assembly-free approach that utilizes ultra-long Oxford Nanopore reads to infer the length distribution of satellite repeat arrays, their association with other repeats and the prevailing sequence periodicities. Using the satellite DNA-rich legume plant Lathyrus sativus as a model, we demonstrated this approach by analyzing 11 major satellite repeats using a set of nanopore reads ranging from 30 to over 200 kb in length and representing 0.73× genome coverage. We found surprising differences between the analyzed repeats because only two of them were predominantly organized in long arrays typical for satellite DNA. The remaining nine satellites were found to be derived from short tandem arrays located within LTR-retrotransposons that occasionally expanded in length. While the corresponding LTR-retrotransposons were dispersed across the genome, this array expansion occurred mainly in the primary constrictions of the L. sativus chromosomes, which suggests that these genome regions are favourable for satellite DNA accumulation.

• Keywords
• Lathyrus sativus, centromeres, fluorescence in situ hybridization (FISH), heterochromatin, long-range organization, nanopore sequencing, satellite DNA, sequence evolution, technical advance,
• MeSH
• Centromere MeSH
• Chromosomes, Plant MeSH
• DNA, Plant genetics   MeSH
• Gene Frequency * MeSH
• Genome, Plant MeSH
• Heterochromatin MeSH
• Lathyrus genetics   MeSH
• Evolution, Molecular MeSH
• Nanopores * MeSH
• Retroelements * MeSH
• DNA, Satellite * MeSH
• Tandem Repeat Sequences * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA, Plant MeSH
• Heterochromatin MeSH
• Retroelements * MeSH
• DNA, Satellite * MeSH

Satellite DNA, a class of repetitive sequences forming long arrays of tandemly repeated units, represents substantial portions of many plant genomes yet remains poorly characterized due to various methodological obstacles. Here we show that the genome of the field bean (Vicia faba, 2n = 12), a long-established model for cytogenetic studies in plants, contains a diverse set of satellite repeats, most of which remained concealed until their present investigation. Using next-generation sequencing combined with novel bioinformatics tools, we reconstructed consensus sequences of 23 novel satellite repeats representing 0.008-2.700% of the genome and mapped their distribution on chromosomes. We found that in addition to typical satellites with monomers hundreds of nucleotides long, V. faba contains a large number of satellite repeats with unusually long monomers (687-2033 bp), which are predominantly localized in pericentromeric regions. Using chromatin immunoprecipitation with CenH3 antibody, we revealed an extraordinary diversity of centromeric satellites, consisting of seven repeats with chromosome-specific distribution. We also found that in spite of their different nucleotide sequences, all centromeric repeats are replicated during mid-S phase, while most other satellites are replicated in the first part of late S phase, followed by a single family of FokI repeats representing the latest replicating chromatin.

• MeSH
• Molecular Sequence Annotation MeSH
• Centromere metabolism   MeSH
• Chromatin Immunoprecipitation MeSH
• DNA, Plant genetics   metabolism   MeSH
• Genome, Plant genetics   MeSH
• Chromosome Mapping methods   MeSH
• Evolution, Molecular MeSH
• DNA Replication Timing genetics   MeSH
• DNA, Satellite genetics   MeSH
• Sequence Analysis, DNA MeSH
• Vicia faba genetics   metabolism   MeSH
• Computational Biology MeSH
• High-Throughput Nucleotide Sequencing MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA, Plant MeSH
• DNA, Satellite MeSH

BACKGROUND: Pikes represent an important genus (Esox) harbouring a pre-duplication karyotype (2n = 2x = 50) of economically important salmonid pseudopolyploids. Here, we have characterized the 5S ribosomal RNA genes (rDNA) in Esox lucius and its closely related E. cisalpinus using cytogenetic, molecular and genomic approaches. Intragenomic homogeneity and copy number estimation was carried out using Illumina reads. The higher-order structure of rDNA arrays was investigated by the analysis of long PacBio reads. Position of loci on chromosomes was determined by FISH. DNA methylation was analysed by methylation-sensitive restriction enzymes. RESULTS: The 5S rDNA loci occupy exclusively (peri)centromeric regions on 30-38 acrocentric chromosomes in both E. lucius and E. cisalpinus. The large number of loci is accompanied by extreme amplification of genes (>20,000 copies), which is to the best of our knowledge one of the highest copy number of rRNA genes in animals ever reported. Conserved secondary structures of predicted 5S rRNAs indicate that most of the amplified genes are potentially functional. Only few SNPs were found in genic regions indicating their high homogeneity while intergenic spacers were more heterogeneous and several families were identified. Analysis of 10-30 kb-long molecules sequenced by the PacBio technology (containing about 40% of total 5S rDNA) revealed that the vast majority (96%) of genes are organised in large several kilobase-long blocks. Dispersed genes or short tandems were less common (4%). The adjacent 5S blocks were directly linked, separated by intervening DNA and even inverted. The 5S units differing in the intergenic spacers formed both homogeneous and heterogeneous (mixed) blocks indicating variable degree of homogenisation between the loci. Both E. lucius and E. cisalpinus 5S rDNA was heavily methylated at CG dinucleotides. CONCLUSIONS: Extreme amplification of 5S rRNA genes in the Esox genome occurred in the absence of significant pseudogenisation suggesting its recent origin and/or intensive homogenisation processes. The dense methylation of units indicates that powerful epigenetic mechanisms have evolved in this group of fish to silence amplified genes. We discuss how the higher-order repeat structures impact on homogenisation of 5S rDNA in the genome.

• Keywords
• Chromosome, Esox, Evolution, Fish, Single cell PacBio sequencing, rDNA,
• MeSH
• Esocidae genetics   MeSH
• Phylogeny MeSH
• Genetic Loci genetics   MeSH
• Genomics * MeSH
• Gene Dosage MeSH
• Heterochromatin metabolism   MeSH
• Conserved Sequence MeSH
• DNA Methylation * MeSH
• DNA, Ribosomal genetics   MeSH
• Base Sequence MeSH
• Oligonucleotide Array Sequence Analysis MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Heterochromatin MeSH
• DNA, Ribosomal MeSH

BACKGROUND AND AIMS: Brassica napus (AACC, 2n = 38, oilseed rape) is a relatively recent allotetraploid species derived from the putative progenitor diploid species Brassica rapa (AA, 2n = 20) and Brassica oleracea (CC, 2n = 18). To determine the influence of intensive breeding conditions on the evolution of its genome, we analysed structure and copy number of rDNA in 21 cultivars of B. napus, representative of genetic diversity. METHODS: We used next-generation sequencing genomic approaches, Southern blot hybridization, expression analysis and fluorescence in situ hybridization (FISH). Subgenome-specific sequences derived from rDNA intergenic spacers (IGS) were used as probes for identification of loci composition on chromosomes. KEY RESULTS: Most B. napus cultivars (18/21, 86 %) had more A-genome than C-genome rDNA copies. Three cultivars analysed by FISH ('Darmor', 'Yudal' and 'Asparagus kale') harboured the same number (12 per diploid set) of loci. In B. napus 'Darmor', the A-genome-specific rDNA probe hybridized to all 12 rDNA loci (eight on the A-genome and four on the C-genome) while the C-genome-specific probe showed weak signals on the C-genome loci only. Deep sequencing revealed high homogeneity of arrays suggesting that the C-genome genes were largely overwritten by the A-genome variants in B. napus 'Darmor'. In contrast, B. napus 'Yudal' showed a lack of gene conversion evidenced by additive inheritance of progenitor rDNA variants and highly localized hybridization signals of subgenome-specific probes on chromosomes. Brassica napus 'Asparagus kale' showed an intermediate pattern to 'Darmor' and 'Yudal'. At the expression level, most cultivars (95 %) exhibited stable A-genome nucleolar dominance while one cultivar ('Norin 9') showed co-dominance. CONCLUSIONS: The B. napus cultivars differ in the degree and direction of rDNA homogenization. The prevalent direction of gene conversion (towards the A-genome) correlates with the direction of expression dominance indicating that gene activity may be needed for interlocus gene conversion.

• Keywords
• Brassica napus, allopolyploidy, chromosome evolution, gene conversion, rDNA,
• MeSH
• Brassica napus genetics   MeSH
• Genetic Variation genetics   MeSH
• Genetic Loci genetics   MeSH
• Gene Conversion genetics   MeSH
• In Situ Hybridization, Fluorescence MeSH
• DNA, Ribosomal genetics   MeSH
• Blotting, Southern MeSH
• Gene Expression Profiling MeSH
• High-Throughput Nucleotide Sequencing MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA, Ribosomal MeSH

BACKGROUND AND AIMS: Chromosomal evolution, including numerical and structural changes, is a major force in plant diversification and speciation. This study addresses genomic changes associated with the extensive chromosomal variation of the Mediterranean Prospero autumnale complex (Hyacinthaceae), which includes four diploid cytotypes each with a unique combination of chromosome number (x = 5, 6, 7), rDNA loci and genome size. METHODS: A new satellite repeat PaB6 has previously been identified, and monomers were reconstructed from next-generation sequencing (NGS) data of P. autumnale cytotype B(6)B(6) (2n = 12). Monomers of all other Prospero cytotypes and species were sequenced to check for lineage-specific mutations. Copy number, restriction patterns and methylation levels of PaB6 were analysed using Southern blotting. PaB6 was localized on chromosomes using fluorescence in situ hybridization (FISH). KEY RESULTS: The monomer of PaB6 is 249 bp long, contains several intact and truncated vertebrate-type telomeric repeats and is highly methylated. PaB6 is exceptional because of its high copy number and unprecedented variation among diploid cytotypes, ranging from 10(4) to 10(6) copies per 1C. PaB6 is always located in pericentromeric regions of several to all chromosomes. Additionally, two lineages of cytotype B(7)B(7) (x = 7), possessing either a single or duplicated 5S rDNA locus, differ in PaB6 copy number; the ancestral condition of a single locus is associated with higher PaB6 copy numbers. CONCLUSIONS: Although present in all Prospero species, PaB6 has undergone differential amplification only in chromosomally variable P. autumnale, particularly in cytotypes B(6)B(6) and B(5)B(5). These arose via independent chromosomal fusions from x = 7 to x = 6 and 5, respectively, accompanied by genome size increases. The copy numbers of satellite DNA PaB6 are among the highest in angiosperms, and changes of PaB6 are exceptionally dynamic in this group of closely related cytotypes of a single species. The evolution of the PaB6 copy numbers is discussed, and it is suggested that PaB6 represents a recent and highly dynamic system originating from a small pool of ancestral repeats.

• Keywords
• Hyacinthaceae, PaB6, Prospero autumnale, chromosomal evolution, copy number, differential amplification, fluorescence in situ hybridization (FISH), genome size, next-generation sequencing, pericentric satellite DNA,
• MeSH
• Chromosomes, Plant genetics   MeSH
• Diploidy MeSH
• DNA, Plant genetics   MeSH
• Phylogeny MeSH
• Genome, Plant MeSH
• Liliaceae genetics   MeSH
• Models, Genetic MeSH
• Evolution, Molecular MeSH
• Molecular Sequence Data MeSH
• Polymerase Chain Reaction * MeSH
• Repetitive Sequences, Nucleic Acid genetics   MeSH
• DNA, Satellite genetics   MeSH
• Base Sequence MeSH
• Sequence Analysis, DNA MeSH
• Telomere metabolism   MeSH
• DNA Copy Number Variations MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA, Plant MeSH
• DNA, Satellite MeSH

Satellite DNA sequences consist of tandemly arranged repetitive units up to thousands nucleotides long in head-to-tail orientation. The evolutionary processes by which satellites arise and evolve include unequal crossing over, gene conversion, transposition and extra chromosomal circular DNA formation. Large blocks of satellite DNA are often observed in heterochromatic regions of chromosomes and are a typical component of centromeric and telomeric regions. Satellite-rich loci may show specific banding patterns and facilitate chromosome identification and analysis of structural chromosome changes. Unlike many other genomes, nuclear genomes of banana (Musa spp.) are poor in satellite DNA and the information on this class of DNA remains limited. The banana cultivars are seed sterile clones originating mostly from natural intra-specific crosses within M. acuminata (A genome) and inter-specific crosses between M. acuminata and M. balbisiana (B genome). Previous studies revealed the closely related nature of the A and B genomes, including similarities in repetitive DNA. In this study we focused on two main banana DNA satellites, which were previously identified in silico. Their genomic organization and molecular diversity was analyzed in a set of nineteen Musa accessions, including representatives of A, B and S (M. schizocarpa) genomes and their inter-specific hybrids. The two DNA satellites showed a high level of sequence conservation within, and a high homology between Musa species. FISH with probes for the satellite DNA sequences, rRNA genes and a single-copy BAC clone 2G17 resulted in characteristic chromosome banding patterns in M. acuminata and M. balbisiana which may aid in determining genomic constitution in interspecific hybrids. In addition to improving the knowledge on Musa satellite DNA, our study increases the number of cytogenetic markers and the number of individual chromosomes, which can be identified in Musa.

• MeSH
• Musa genetics   MeSH
• Chromosomes, Plant MeSH
• Diploidy MeSH
• Phylogeny MeSH
• Genetic Variation MeSH
• Genome, Plant * MeSH
• Chromosome Mapping MeSH
• Molecular Sequence Data MeSH
• Genes, Plant MeSH
• DNA, Satellite * MeSH
• Base Sequence MeSH
• Sequence Analysis, DNA MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA, Satellite * MeSH

Chromatin Assembly Factor 1 (CAF1) is a three-subunit H3/H4 histone chaperone responsible for replication-dependent nucleosome assembly. It is composed of CAC 1-3 in yeast; p155, p60, and p48 in humans; and FASCIATA1 (FAS1), FAS2, and MULTICOPY SUPPRESSOR OF IRA1 in Arabidopsis thaliana. We report that disruption of CAF1 function by fas mutations in Arabidopsis results in telomere shortening and loss of 45S rDNA, while other repetitive sequences (5S rDNA, centromeric 180-bp repeat, CACTA, and Athila) are unaffected. Substantial telomere shortening occurs immediately after the loss of functional CAF1 and slows down at telomeres shortened to median lengths around 1 to 1.5 kb. The 45S rDNA loss is progressive, leaving 10 to 15% of the original number of repeats in the 5th generation of mutants affecting CAF1, but the level of the 45S rRNA transcripts is not altered in these mutants. Increasing severity of the fas phenotype is accompanied by accumulation of anaphase bridges, reduced viability, and plant sterility. Our results show that appropriate replication-dependent chromatin assembly is specifically required for stable maintenance of telomeres and 45S rDNA.

• MeSH
• Arabidopsis genetics   metabolism   MeSH
• DNA, Plant genetics   metabolism   MeSH
• Chromatin Assembly Factor-1 genetics   metabolism   MeSH
• Mutagenesis, Insertional MeSH
• Mutation MeSH
• Arabidopsis Proteins genetics   metabolism   MeSH
• Gene Expression Regulation, Plant MeSH
• Chromatin Assembly and Disassembly MeSH
• DNA, Ribosomal genetics   metabolism   MeSH
• RNA, Ribosomal genetics   metabolism   MeSH
• Telomere metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA, Plant MeSH
• Chromatin Assembly Factor-1 MeSH
• FAS protein, Arabidopsis MeSH Browser
• Arabidopsis Proteins MeSH
• DNA, Ribosomal MeSH
• RNA, Ribosomal MeSH
• RNA, ribosomal, 45S MeSH Browser