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Eukaryotes have varying numbers and structures of characteristic chromosomes across lineages or species. The evolutionary trajectory of species may have been affected by spontaneous genome rearrangements. Chromosome fusion drastically alters karyotypes. However, the mechanisms and consequences of chromosome fusions, particularly in muntjac species, are poorly understood. Recent research-based advancements in three-dimensional (3D) genomics, particularly high-throughput chromatin conformation capture (Hi-C) sequencing, have allowed for the identification of chromosome fusions and provided mechanistic insights into three muntjac species: Muntiacus muntjak, M. reevesi, and M. crinifrons. This study aimed to uncover potential genome rearrangement patterns in the threatened species Fea’s muntjac (Muntiacus feae), which have not been previously examined for such characteristics. Deep Hi-C sequencing (31.42 × coverage) was performed to reveal the 3D chromatin architecture of the Fea’s muntjac genome. Patterns of repeated chromosome fusions that were potentially mediated by high-abundance transposable elements were identified. Comparative Hi-C maps demonstrated linkage homology between the sex chromosomes in Fea’s muntjac and autosomes in M. reevesi, indicating that fusions may have played a crucial role in the evolution of the sex chromosomes of the lineage. The species-level dynamics of topologically associated domains (TADs) suggest that TAD organization could be altered by differential chromosome interactions owing to repeated chromosome fusions. However, research on the effect of TADs on muntjac genome evolution is insufficient. This study generated Hi-C data for the Fea’s muntjac, providing a genomic resource for future investigations of the evolutionary patterns of chromatin conformation at the chromosomal level. [ABSTRACT FROM AUTHOR]

Abstract Background Experimental proof of gene function assignments in plants is based on mutant analyses. T-DNA insertion lines provided an invaluable resource of mutants and enabled systematic reverse genetics-based investigation of the functions of Arabidopsis thaliana genes during the last decades. Results We sequenced the genomes of 14 A. thaliana GABI-Kat T-DNA insertion lines, which eluded flanking sequence tag-based attempts to characterize their insertion loci, with Oxford Nanopore Technologies (ONT) long reads. Complex T-DNA insertions were resolved and 11 previously unknown T-DNA loci identified, resulting in about 2 T-DNA insertions per line and suggesting that this number was previously underestimated. T-DNA mutagenesis caused fusions of chromosomes along with compensating translocations to keep the gene set complete throughout meiosis. Also, an inverted duplication of 800 kbp was detected. About 10 % of GABI-Kat lines might be affected by chromosomal rearrangements, some of which do not involve T-DNA. Local assembly of selected reads was shown to be a computationally effective method to resolve the structure of T-DNA insertion loci. We developed an automated workflow to support investigation of long read data from T-DNA insertion lines. All steps from DNA extraction to assembly of T-DNA loci can be completed within days. Conclusions Long read sequencing was demonstrated to be an effective way to resolve complex T-DNA insertions and chromosome fusions. Many T-DNA insertions comprise not just a single T-DNA, but complex arrays of multiple T-DNAs. It is becoming obvious that T-DNA insertion alleles must be characterized by exact identification of both T-DNA::genome junctions to generate clear genotype-to-phenotype relations.

The African weakly electric elephantfish family Mormyridae comprises 22 genera and almost 230 species. Up-to-date cytogenetic information was available for 17 species representing 14 genera. Here we report chromosome number and morphology in Hyperopisus bebe (Lacepède, 1803) and Pollimyrus isidori (Valenciennes, 1847) collected from the White Nile system in southwestern Ethiopia. Both taxa displayed the diploid chromosome number 2n = 40, but they differed in fundamental numbers: FN = 66 in H. bebe and FN = 72 in P. isidori; previously the same diploid chromosome number 2n = 40 was reported in an undescribed species of Pollimyrus Taverne, 1971 (FN = 42) from the same region. Our results demonstrate that not only pericentric inversions, but fusions also played a substantial role in the evolution of the mormyrid karyotype structure. If the hypothesis that the karyotype structure with 2n = 50-52 and prevalence of the uni-armed chromosomes close to the ancestral condition for the family Mormyridae is correct, the most derived karyotype structures are found in the Mormyrus Linnaeus, 1758 species with 2n = 50 and the highest number of bi-armed elements in their compliments compared to all other mormyrids and in Pollimyrus isidori with the highest number of bi-armed elements among the mormyrids with 2n = 40. [ABSTRACT FROM AUTHOR]

Background: Experimental proof of gene function assignments in plants is based on mutant analyses. T-DNA insertion lines provided an invaluable resource of mutants and enabled systematic reverse genetics-based investigation of the functions of Arabidopsis thaliana genes during the last decades. Results: We sequenced the genomes of 14 A. thaliana GABI-Kat T-DNA insertion lines, which eluded flanking sequence tag-based attempts to characterize their insertion loci, with Oxford Nanopore Technologies (ONT) long reads. Complex T-DNA insertions were resolved and 11 previously unknown T-DNA loci identified, resulting in about 2 T-DNA insertions per line and suggesting that this number was previously underestimated. T-DNA mutagenesis caused fusions of chromosomes along with compensating translocations to keep the gene set complete throughout meiosis. Also, an inverted duplication of 800 kbp was detected. About 10 % of GABI-Kat lines might be affected by chromosomal rearrangements, some of which do not involve T-DNA. Local assembly of selected reads was shown to be a computationally effective method to resolve the structure of T-DNA insertion loci. We developed an automated workflow to support investigation of long read data from T-DNA insertion lines. All steps from DNA extraction to assembly of T-DNA loci can be completed within days. Conclusions: Long read sequencing was demonstrated to be an effective way to resolve complex T-DNA insertions and chromosome fusions. Many T-DNA insertions comprise not just a single T-DNA, but complex arrays of multiple T-DNAs. It is becoming obvious that T-DNA insertion alleles must be characterized by exact identification of both T-DNA::genome junctions to generate clear genotype-to-phenotype relations. [ABSTRACT FROM AUTHOR]

Telomere dysfunction plays a complex role in tumorigenesis. While dysfunctional telomeres can block the proliferation of incipient cancer clones by inducing replicative senescence, fusion of dysfunctional telomeres can drive genome instability and oncogenic genomic rearrangements. Therefore, it is important to define the regulatory pathways that guide these opposing effects. Recent work has shown that the autophagy pathway regulates both senescence and genome instability in various contexts. Here, we apply models of acute telomere dysfunction to determine whether autophagy modulates the resulting genome instability and senescence responses. While telomere dysfunction rapidly induces autophagic flux in human fibroblast cell lines, inhibition of the autophagy pathway does not have a significant impact upon the transition to senescence, in contrast to what has previously been reported for oncogene-induced senescence. Our results suggest that this difference may be explained by disparities in the development of the senescence-associated secretory phenotype. We also show that chromosome fusions induced by telomere dysfunction are comparable in autophagy-proficient and autophagy-deficient cells. Altogether, our results highlight the complexity of the senescence-autophagy interface and indicate that autophagy induction is unlikely to play a significant role in telomere dysfunction-driven senescence and chromosome fusions.

Repressor/activator protein 1 (RAP1) is a highly evolutionarily conserved protein found at telomeres. Although yeast Rap1 is a key telomere capping protein preventing non‐homologous end joining (NHEJ) and consequently telomere fusions, its role at mammalian telomeres in vivo is still controversial. Here, we demonstrate that RAP1 is required to protect telomeres in replicative senescent human cells. Downregulation of RAP1 in these cells, but not in young or dividing pre‐senescent cells, leads to telomere uncapping and fusions. The anti‐fusion effect of RAP1 was further explored in a HeLa cell line where RAP1 expression was depleted through an inducible CRISPR/Cas9 strategy. Depletion of RAP1 in these cells gives rise to telomere fusions only when telomerase is inhibited. We further show that the fusions triggered by RAP1 loss are dependent upon DNA ligase IV. We conclude that human RAP1 is specifically involved in protecting critically short telomeres. This has important implications for the functions of telomeres in senescent cells. Synopsis: Human RAP1 specifically protects critically short telomeres from DNA damage response activation and telomere fusions in replicative senescent fibroblasts and tumor cells upon telomerase inhibition. RAP1 protects critically short telomeres of replicative senescent cells and HeLa cells upon telomerase inhibition.Telomere fusions generated by RAP1 depletion, are dependent on DNA ligase IV.RAP1 downregulation compromises re‐growth of replicative senescent cells upon p21 inhibition. [ABSTRACT FROM AUTHOR]

The Heliconius butterflies are a widely studied adaptive radiation of 46 species spread across Central and South America, several of which are known to hybridize in the wild. Here, we present a substantially improved assembly of the Heliconius melpomene genome, developed using novel methods that should be applicable to improving other genome assemblies produced using short read sequencing. First, we whole-genome-sequenced a pedigree to produce a linkage map incorporating 99% of the genome. Second, we incorporated haplotype scaffolds extensively to produce a more complete haploid version of the draft genome. Third, we incorporated ∼20x coverage of Pacific Biosciences sequencing, and scaffolded the haploid genome using an assembly of this long-read sequence. These improvements result in a genome of 795 scaffolds, 275 Mb in length, with an N50 length of 2.1 Mb, an N50 number of 34, and with 99% of the genome placed, and 84% anchored on chromosomes. We use the new genome assembly to confirm that the Heliconius genome underwent 10 chromosome fusions since the split with its sister genus Eueides, over a period of about 6 million yr.

The Heliconius butterflies are a widely studied adaptive radiation of 46 species spread across Central and South America, several of which are known to hybridize in the wild. Here, we present a substantially improved assembly of the Heliconius melpomene genome, developed using novel methods that should be applicable to improving other genome assemblies produced using short read sequencing. First, we wholegenome- sequenced a pedigree to produce a linkage map incorporating 99% of the genome. Second, we incorporated haplotype scaffolds extensively to produce a more complete haploid version of the draft genome. Third, we incorporated ~20x coverage of Pacific Biosciences sequencing, and scaffolded the haploid genome using an assembly of this long-read sequence. These improvements result in a genome of 795 scaffolds, 275 Mb in length, with an N50 length of 2.1 Mb, an N50 number of 34, and with 99% of the genome placed, and 84% anchored on chromosomes. We use the new genome assembly to confirm that the Heliconius genome underwent 10 chromosome fusions since the split with its sister genus Eueides, over a period of about 6 million yr. [ABSTRACT FROM AUTHOR]

Phleum echinatum Host (2 n = 2 x = 10) is an annual Mediterranean species which differs from other representatives of the genus Phleum by reduced chromosome number, asymmetric karyotype and unusually high amount of DNA in the genome. Chromosomes of this plant were studied using conventional acetic-orcein staining and fluorescence in situ hybridization (FISH). FISH showed the major 35S ribosomal DNA (rDNA) site at the secondary constriction of satellite chromosome (3) and the minor 35S rDNA site near 5S rDNA cluster in the monobrachial chromosome 5. Telomeric repeats were detected at all chromosome ends within secondary constriction in satellited chromosome 3 and at the centromeric regions of chromosomes 1 and 2. Intrachromosomally located telomeric repeats are probably traces of chromosomal rearrangements that have shaped P.echinatum genome; they were prone to breakage which was manifested in chromosome fragmentation. The most distinct telomeric signals, suggesting massive amplification of interstitial telomeric sequences (ITRs), were observed at the nucleolar organizer region (NOR) of the third chromosome pair. Double FISH confirmed co-localization of telomeric and 35S rDNA repeats in this locus characterized by the biggest fragility in the karyotype. Fragile sites of P.echinatum, composed of amplified telomeric repeats, may bear a resemblance to metazoan rare fragile sites enriched in microsatellite repeats. [ABSTRACT FROM AUTHOR]

In fishes, as in other vertebrate species, the DNA component of the telomeres consists of the tandemly repeated TTAGGG motif. The length of the telomeric arrays in fishes ranges from 2 to 25 kb and shortens with age in some of the species. To date, chromosomal distribution of the telomeric DNA sequences has been examined in approximately 80 fish species of which about 42% show additional telomeric hybridization signals far from the chromosomal termini. Based on the chromosomal location, such internally located telomeric repeats may be classified into 4 categories: (1) telomeric DNA sequences located at the pericentromeric regions, (2) interstitial telomeric sites observed between centromeres and the bona fide telomeres, (3) telomeric DNA sequences that scatter along the nucleolus organizer regions, and (4) telomeric DNA repeats interspersed with the entire chromosomes. Most of the pericentromeric and interstitial telomeric sequences in fish are possible relicts of chromosome fusion events. The origin of the telomeric sequences co- localizing with the major rDNA sequences or scattered along the whole chromosomes is not clear. Internally located telomeric repeats are considered as 'hot spots' for recombination and thus may potentially increase the rates of chromosome breaks and rearrangements leading to the various chromosomal polymorphisms in fishes. FISH with telomeric probe applied to metaphase spreads of androgenetic specimens that hatched from eggs exposed to ionizing radiation before insemination enabled the detection of small radiation-induced fragments of maternal chromosomes. Remnants of the irradiated chromosomes were found to be ring chromosomes with the interstitial telomeric signals, telomerless rings, fragments with fused sister chromatids, and linear fragments with telomeres detected at both of their ends. The increasing availability of techniques enabling the study of fish telomeres and telomerase and the easy access to numerous fish species strongly confirm that these animals are promising models in research concerning the role of telomeres and telomerase in vertebrate aging, repair of ionizing radiation-induced DNA double strand breaks, and chromosomal rearrangements. © 2013 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Here we report the consequences of telomere erosion in Arabidopsis thaliana, studied by examining seed and pollen production and the course of male meiosis through the last five generations (G5-G9) of telomerase-deficient Arabidopsis mutants. We used a previously described mutant line in which telomerase activity was abolished by T-DNA insertion into the TERT gene encoding telomerase reverse transcriptase. Reduced fertility accompanied by morphological abnormalities occurred in G6, which produced on average 35 seeds per silique (vs. 43 in wild type) and worsened in G7 (30 seeds) and G8 (14 seeds), as did the morphological abnormalities. The last generation of tert mutants (G9) did not form reproductive organs. Analysis of meiosis indicated that the main cause of reduced fertility in the late generation tert mutants of Arabidopsis was the numerous chromosomal end-to-end fusions which led to massive genome rearrangements in meiocytes. Fusion of meiotic chromosomes began in G5 and increased in each of the next generations. Unpaired chromosomes (univalents) were observed in G7 and G8. The study highlights some differences in the meiotic consequences of telomere shortening between plant and animal systems. [ABSTRACT FROM AUTHOR]

Here we provide the first detailed description of the hybrid zone between the Cremona chromosomal race of house mouse (ICRE; 2n = 22) and the standard all-telocentric race (40ST; 2n = 40), with full karyotypes of 106 individuals from 17 localities along a transect between the 2 races to the west of Lake Garda in Northern Italy. The ICRE race is characterised by 9 pairs of metacentric chromosomes in a homozygous state and we use the metacentric frequency data along the transect to fit tanh metacentric clines. The clines are narrow (5-8 km, standardised width) suggesting low hybrid fitness. However, the lack of occurrence of ICRE × 40ST F1 hybrids and presence of other hybrid types suggests that the F1 hybrids initially produced in this hybrid zone were at least partially fertile, despite having 9 meiotic trivalent configurations. We apply the same cline-fitting methodology to 3 previously studied hybrid zones between metacentric races and the 40ST race. Taken together with published clinal data on 4 further metacentric-40ST hybrid zones, we are able to make objective generalisations on the characteristics of such zones in the house mouse. Zones involving 22-chromosome races are narrower, on average, than other metacentric-40ST hybrid zones and do not show a tendency towards the generation of new races as found with zones where the metacentric race has a higher 2n. It appears that metacentric-40ST zones are unlikely to be sites of speciation (even when a 22-chromosome race is involved), although a mosaic structure to the hybrid zone may enhance this possibility. We make a comparison between metacentric-40ST zones and contacts between 2 metacentric races, for a comprehensive perspective of chromosomal hybrid zones in the house mouse. Copyright © 2011 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Although the standard karyotype of the western house mouse (Mus musculus domesticus) consists entirely of telocentric chromosomes, there are over 100 populations across western Europe and North Africa characterized by different sets of metacentrics formed by Robertsonian fusions and whole-arm reciprocal translocations. Here we report the discovery of a new metacentric population from Valchiavenna, northern Italy, that we have named the ‘Lower Valchiavenna population’ (abbreviated as ILVC). This metacentric population is found in villages and on farms over a 10-kilometer stretch comprising the southern half of Valchiavenna. ILVC is characterized by the metacentrics 1.18, 2.4, 3.8, 5.15, 6.7, 9.14, 10.12, 11.13 and 16.17, and appears to be closely related to the Chiasso population (CHCH), which possesses the same set of metacentrics except 1.18. We discuss the evolutionary origin of ILVC in relation to human occupation of the region. We also suggest that the geographical position of ILVC between 2 other metacentric populations with entirely different sets of metacentrics (Chiavenna, ICHI, and lower Valtellina, ILVA) may provide 2 additional chromosomal hybrid zones for the study of speciation. Copyright © 2009 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

DNA-PKcs is the catalytic subunit of the DNA- dependent protein kinase (DNA-PK) complex that functions in the non-homologous end-joining of double-strand breaks, and it has been shown previously to have a role in telomere capping. In particular, DNA-PKcs deficiency leads to chromosome fusions involving telomeres produced by leading-strand synthesis. Here, by generating mice doubly deficient in DNA-PKcs and telomerase (Terc-/-/DNA-PKcs-/-), we demonstrate that DNA-PKcs also has a fundamental role in telomere length maintenance. In particular, Terc-/-/DNA-PKcs-/- mice displayed an accelerated rate of telomere shortening when compared with Terc-/- controls, suggesting a functional interaction between both activities in maintaining telomere length. In addition, we also provide direct demonstration that DNA-PKcs is essential for both end-to-end fusions and apoptosis triggered by critically short telomeres. Our data predict that, in telomerase-deficient cells, i.e. human somatic cells, DNA-PKcs abrogation may lead to a faster rate of telomere degradation and cell cycle arrest in the absence of increased apoptosis and/or fusion of telomere-exhausted chromosomes. These results suggest a critical role of DNA-PKcs in both cancer and aging. [ABSTRACT FROM AUTHOR]

Telomeres, the complex nucleoprotein structures at the ends of linear eukaryotic chromosomes, along with telomerase, the enzyme that synthesizes telomeric DNA, are required to maintain a stable genome. Together, the enzyme and substrate perform this essential service by protecting chromosomes from exonucleolytic degradation and end-to-end fusions and by compensating for the inability of conventional DNA replication machinery to completely duplicate the ends of linear chromosomes. Telomeres are also important for chromosome organization within the nucleus, especially during mitosis and meiosis. The contributions of telomeres and telomerases to plant genome stability have been confirmed by analysis of Arabidopsis mutants that lack telomerase activity. These mutants have unstable genomes, but manage to survive up to ten generations with increasingly shortened telomeres and cytogenetic abnormalities. Comparisons between telomerase-deficient Arabidopsis and telomerase-deficient mice reveal distinct differences in the consequences of massive genome damage, probably reflecting the greater developmental and genomic plasticity of plants. [ABSTRACT FROM AUTHOR]