Hyperextended telomeres promote formation of C-circle DNA in telomerase positive human cells.

Telomere length maintenance is crucial to cancer cell immortality. Up to 15% of cancers utilize a telomerase-independent, recombination-based mechanism termed alternative lengthening of telomeres (ALT). Currently, the primary ALT biomarker is the C-circle, a type of circular DNA with extrachromosomal telomere repeats (cECTRs). How C-circles form is not well characterized. We investigated C-circle formation in the human cen3tel cell line, a long-telomere, telomerase+ (LTT+) cell line with progressively hyper-elongated telomeres (up to ∼100 kb). cECTR signal was observed in 2D gels and C-circle assays but not t-circle assays, which also detect circular DNA with extrachromosomal telomere repeats. Telomerase activity and C-circle signal were not separable in the analysis of clonal populations, consistent with C-circle production occurring within telomerase+ cells. We observed similar cECTR results in two other LTT+ cell lines, HeLa1.3 (∼23 kb telomeres) and HeLaE1 (∼50 kb telomeres). In LTT+ cells, telomerase activity did not directly impact C-circle signal; instead, C-circle signal correlated with telomere length. LTT+ cell lines were less sensitive to hydroxyurea than ALT+ cell lines, suggesting that ALT status is a stronger contributor to replication stress levels than telomere length. Additionally, the DNA repair-associated protein FANCM did not suppress C-circles in LTT+ cells as it does in ALT+ cells. Thus, C-circle formation may be driven by telomere length, independently of telomerase and replication stress, highlighting limitations of C-circles as a stand-alone ALT biomarker.

Prospects of telomere-to-telomere assembly in barley: Analysis of sequence gaps in the MorexV3 reference genome.

The first gapless, telomere-to-telomere (T2T) sequence assemblies of plant chromosomes were reported recently. However, sequence assemblies of most plant genomes remain fragmented. Only recent breakthroughs in accurate long-read sequencing have made it possible to achieve highly contiguous sequence assemblies with a few tens of contigs per chromosome, that is a number small enough to allow for a systematic inquiry into the causes of the remaining sequence gaps and the approaches and resources needed to close them. Here, we analyse sequence gaps in the current reference genome sequence of barley cv. Morex (MorexV3). Optical map and sequence raw data, complemented by ChIP-seq data for centromeric histone variant CENH3, were used to estimate the abundance of centromeric, ribosomal DNA, and subtelomeric repeats in the barley genome. These estimates were compared with copy numbers in the MorexV3 pseudomolecule sequence. We found that almost all centromeric sequences and 45S ribosomal DNA repeat arrays were absent from the MorexV3 pseudomolecules and that the majority of sequence gaps can be attributed to assembly breakdown in long stretches of satellite repeats. However, missing sequences cannot fully account for the difference between assembly size and flow cytometric genome size estimates. We discuss the prospects of gap closure with ultra-long sequence reads.

Telomerase detection using a DNA-PAINT strategy.

Telomerase plays an important role in maintaining the length of telomere during cell division and is recognized as a new kind of biomarkers for cancer diagnosis. In this work, we present a brand new telomerase detection strategy based on a DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) like strategy. With an extraordinary spatial resolution (∼10 nm), the DNA-PAINT based strategy offers several advantages. First, it avoids complicated polymerase chain reaction and electrophoresis procedures. Second, it enables super resolution imaging of the reaction products with a high signal-to-noise ratio and facilitates the location of telomeric elongation sites on the single particle level, which results in a high sensitivity. Third, the detection scheme of the DNA-PAINT strategy allows directin situvisualization of the telomeric elongation process, which has never been achieved before. All these advantages make the DNA-PAINT telomerase detection strategy significant for dynamic investigation of telomerase related physiological processes as well as cancer diagnosis.

Telomeric-Like Repeats Flanked by Sequences Retrotranscribed from the Telomerase RNA Inserted at DNA Double-Strand Break Sites during Vertebrate Genome Evolution.

Interstitial telomeric sequences (ITSs) are stretches of telomeric-like repeats located at internal chromosomal sites. We previously demonstrated that ITSs have been inserted during the repair of DNA double-strand breaks in the course of evolution and that some rodent ITSs, called TERC-ITSs, are flanked by fragments retrotranscribed from the telomerase RNA component (TERC). In this work, we carried out an extensive search of TERC-ITSs in 30 vertebrate genomes and identified 41 such loci in 22 species, including in humans and other primates. The fragment retrotranscribed from the TERC RNA varies in different lineages and its sequence seems to be related to the organization of TERC. Through comparative analysis of TERC-ITSs with orthologous empty loci, we demonstrated that, at each locus, the TERC-like sequence and the ITS have been inserted in one step in the course of evolution. Our findings suggest that telomerase participated in a peculiar pathway of DNA double-strand break repair involving retrotranscription of its RNA component and that this mechanism may be active in all vertebrate species. These results add new evidence to the hypothesis that RNA-templated DNA repair mechanisms are active in vertebrate cells.

A Role for Human DNA Polymerase λ in Alternative Lengthening of Telomeres.

Telomerase negative cancer cell types use the Alternative Lengthening of Telomeres (ALT) pathway to elongate telomeres ends. Here, we show that silencing human DNA polymerase (Pol λ) in ALT cells represses ALT activity and induces telomeric stress. In addition, replication stress in the absence of Pol λ, strongly affects the survival of ALT cells. In vitro, Pol λ can promote annealing of even a single G-rich telomeric repeat to its complementary strand and use it to prime DNA synthesis. The noncoding telomeric repeat containing RNA TERRA and replication protein A negatively regulate this activity, while the Protection of Telomeres protein 1 (POT1)/TPP1 heterodimer stimulates Pol λ. Pol λ associates with telomeres and colocalizes with TPP1 in cells. In summary, our data suggest a role of Pol λ in the maintenance of telomeres by the ALT mechanism.

Comparative Study of the Bush Dog (Speothos venaticus) Karyotype and Analysis of Satellite DNA Sequences and Their Chromosome Distribution in Six Species of Canidae.

The bush dog (Speothos venaticus, 2n = 74) is a near threatened species taxonomically classified among South American canids. We revised the bush dog karyotype and performed a comparative sequence analysis of satellite and satellite-like DNAs in 6 canids: the bush dog, domestic dog (Canis familiaris, 2n = 78), grey wolf (C. lupus, 2n = 78), Chinese raccoon dog (Nyctereutes procyonoides procyonoides, 2n = 54+B), red fox (Vulpes vulpes, 2n = 34+B), and arctic fox (V. lagopus, 2n = 48-50) to specify the species position among Canidae. Using FISH with painting and BAC probes, we found that the distribution of canid evolutionarily conserved chromosome segments in the bush dog karyotype is similar to that of the domestic dog and grey wolf. The bush dog karyotype differs by 2 acrocentric chromosome pairs formed by tandem fusions of the canine (29;34) and (26;35) orthologues. An interstitial signal of the telomeric probe was observed in the (26;35) fusion site in the bush dog indicating a recent evolutionary origin of this rearrangement. Sequences and hybridisation patterns of satellite DNAs were compared, and a phylogenetic tree of the 6 canid species was constructed which confirmed the bush dog position close to the wolf-like canids, and apart from the raccoon dog and foxes.

The large genome size variation in the Hesperis clade was shaped by the prevalent proliferation of DNA repeats and rarer genome downsizing.

BACKGROUND AND AIMS: Most crucifer species (Brassicaceae) have small nuclear genomes (mean 1C-value 617 Mb). The species with the largest genomes occur within the monophyletic Hesperis clade (Mandáková et al., Plant Physiology174: 2062-2071; also known as Clade E or Lineage III). Whereas most chromosome numbers in the clade are 6 or 7, monoploid genome sizes vary 16-fold (256-4264 Mb). To get an insight into genome size evolution in the Hesperis clade (~350 species in ~48 genera), we aimed to identify, quantify and localize in situ the repeats from which these genomes are built. We analysed nuclear repeatomes in seven species, covering the phylogenetic and genome size breadth of the clade, by low-pass whole-genome sequencing. METHODS: Genome size was estimated by flow cytometry. Genomic DNA was sequenced on an Illumina sequencer and DNA repeats were identified and quantified using RepeatExplorer; the most abundant repeats were localized on chromosomes by fluorescence in situ hybridization. To evaluate the feasibility of bacterial artificial chromosome (BAC)-based comparative chromosome painting in Hesperis-clade species, BACs of arabidopsis were used as painting probes. KEY RESULTS: Most biennial and perennial species of the Hesperis clade possess unusually large nuclear genomes due to the proliferation of long terminal repeat retrotransposons. The prevalent genome expansion was rarely, but repeatedly, counteracted by purging of transposable elements in ephemeral and annual species. CONCLUSIONS: The most common ancestor of the Hesperis clade has experienced genome upsizing due to transposable element amplification. Further genome size increases, dominating diversification of all Hesperis-clade tribes, contrast with the overall stability of chromosome numbers. In some subclades and species genome downsizing occurred, presumably as an adaptive transition to an annual life cycle. The amplification versus purging of transposable elements and tandem repeats impacted the chromosomal architecture of the Hesperis-clade species.

Molecular cytogenetic analysis reveals the existence of two independent neo-XY sex chromosome systems in Anatolian Pamphagidae grasshoppers.

BACKGROUND: Neo-XY sex chromosome determination is a rare event in short horned grasshoppers, but it appears with unusual frequency in the Pamphagidae family. The neo-Y chromosomes found in several species appear to have undergone heterochromatinization and degradation, but this subject needs to be analyzed in other Pamphagidae species. We perform here karyotyping and molecular cytogenetic analyses in 12 Pamphagidae species from the center of biodiversity of this group in the previously-unstudied Anatolian plateau. RESULTS: The basal karyotype for the Pamphagidae family, consisting of 18 acrocentric autosomes and an acrocentric X chromosome (2n♂ = 19, X0; 2n♀ = 20, XX), was found only in G. adaliae. The karyotype of all other studied species consisted of 16 acrocentric autosomes and a neo-XY sex chromosome system (2n♂♀ = 18, neo-XX♀/neo-XY♂). Two different types of neo-Y chromosomes were found. One of them was typical for three species of the Glyphotmethis genus, and showed a neo-Y chromosome being similar in size to the XR arm of the neo-X, with the addition of two small subproximal interstitial C-blocks. The second type of the neo-Y chromosome was smaller and more heterochromatinized than the XR arm, and was typical for all Nocarodeini species studied. The chromosome distribution of C-positive regions and clusters of ribosomal DNA (rDNA) and telomeric repeats yielded additional information on evolution of these neo-XY systems. CONCLUSION: Most Pamphagidae species in the Anatolian region were found to have neo-XY sex chromosome systems, belonging to two different evolutionary lineages, marked by independent X-autosome fusion events occurred within the Trinchinae and Pamphaginae subfamilies. The high density of species carrying neo-XY systems in the Anatolian region, and the different evolutionary stage for the two lineages found, one being older than the other, indicates that this region has a long history of neo-XY sex chromosome formation.

Probing Human Telomeric DNA and RNA Topology and Ligand Binding in a Cellular Model by Using Responsive Fluorescent Nucleoside Probes.

The development of biophysical systems that enable an understanding of the structure and ligand-binding properties of G-quadruplex (GQ)-forming nucleic acid sequences in cells or models that mimic the cellular environment would be highly beneficial in advancing GQ-directed therapeutic strategies. Herein, the establishment of a biophysical platform to investigate the structure and recognition properties of human telomeric (H-Telo) DNA and RNA repeats in a cell-like confined environment by using conformation-sensitive fluorescent nucleoside probes and a widely used cellular model, bis(2-ethylhexyl) sodium sulfosuccinate reverse micelles (RMs), is described. The 2'-deoxy and ribonucleoside probes, composed of a 5-benzofuran uracil base analogue, faithfully report the aqueous micellar core through changes in their fluorescence properties. The nucleoside probes incorporated into different loops of H-Telo DNA and RNA oligonucleotide repeats are minimally perturbing and photophysically signal the formation of respective GQ structures in both aqueous buffer and RMs. Furthermore, these sensors enable a direct comparison of the binding affinity of a ligand to H-Telo DNA and RNA GQ structures in the bulk and confined environment of RMs. These results demonstrate that this combination of a GQ nucleoside probe and easy-to-handle RMs could provide new opportunities to study and devise screening-compatible assays in a cell-like environment to discover GQ binders of clinical potential.

Distribution of Interstitial Telomeric Sequences in Primates and the Pygmy Tree Shrew (Scandentia).

It has been hypothesized that interstitial telomeric sequences (ITSs), i.e., repeated telomeric DNA sequences found at intrachromosomal sites in many vertebrates, could be correlated to chromosomal rearrangements and plasticity. To test this hypothesis, we hybridized a telomeric PNA probe through FISH on representative species of 2 primate infraorders, Strepsirrhini (Lemur catta, Otolemur garnettii, Nycticebus coucang) and Catarrhini (Erythrocebus patas, Cercopithecus petaurista, Chlorocebus aethiops, Colobus guereza), as well as on 1 species of the order Scandentia, Tupaia minor, used as an outgroup for primates in phylogenetic reconstructions. In almost all primate species analyzed, we found a telomeric pattern only. In Tupaia, the hybridization revealed many bright ITSs on at least 11 chromosome pairs, both biarmed and acrocentric. These ITS signals in Tupaia correspond to fusion points of ancestral human syntenic associations, but are also present in other chromosomes showing synteny to only a single human chromosome. This distribution pattern was compared to that of the heterochromatin regions detected through sequential C-banding performed after FISH. Our results in the analyzed species, compared with literature data on ITSs in primates, allowed us to discuss different mechanisms responsible for the origin and distribution of ITSs, supporting the correlation between rearrangements and ITSs.

No Interstitial Telomeres on Autosomes but Remarkable Amplification of Telomeric Repeats on the W Sex Chromosome in the Sand Lizard (Lacerta agilis).

Telomeres are repeat (TTAGGG) n sequences that form terminal ends of chromosomes and have several functions, such as protecting the coding DNA from erosion at mitosis. Due to chromosomal rearrangements through evolutionary history (e.g., inversions and fusions), telomeric sequences are also found between the centromere and the terminal ends (i.e., at interstitial telomeric sites, ITSs). ITS telomere sequences have been implicated in heritable disease caused by genomic instability of ITS polymorphic variants, both with respect to copy number and sequence. In the sand lizard (Lacerta agilis), we have shown that telomere length is predictive of lifetime fitness in females but not males. To assess whether this sex specific fitness effect could be traced to ITSs differences, we mapped (TTAGGG) n sequences using fluorescence in situ hybridization in fibroblast cells cultured from 4 specimens of known sex. No ITSs could be found on autosomes in either sex. However, females have heterogametic sex chromosomes in sand lizards (ZW, 2n = 38) and the female W chromosome showed degeneration and remarkable (TTAGGG) n amplification, which was absent in the Z chromosomes. This work warrants further research on sex chromosome content, in particular of the degenerate W chromosome, and links to female fitness in sand lizards.

Structural effects of inosine substitution in telomeric DNA quadruplex.

The telomeric DNA, a distal region of eukaryotic chromosome containing guanine-rich repetitive sequence of (TTAGGG)n, has been shown to adopt higher-order structures, specifically G-quadruplexes (G4s). Previous studies have demonstrated the implication of G4 in tumor inhibition through chromosome maintenance and manipulation of oncogene expression featuring their G-rich promoter regions. Besides higher order structures, several regulatory roles are attributed to DNA epigenetic markers. In this work, we investigated how the structural dynamics of a G-quadruplex, formed by the telomeric sequence, is affected by inosine, a prevalent modified nucleotide. We used the standard (TTAGGG)n telomere repeats with guanosine mutated to inosine at each G position. Sequences (GGG)4, (IGG)4, (GIG)4, (GGI)4, (IGI)4, (IIG)4, (GII)4, and (III)4, bridged by TTA linker, are studied using biophysical experiments and molecular modeling. The effects of metal cations in quadruplex folding were explored in both Na+ and K+ containing buffers using CD and UV-melting studies. Our results show that antiparallel quadruplex topology forms with the native sequence (GGG)4 and the terminal modified DNAs (IGG)4 and (GGI)4 in both Na+ and K+ containing buffers. Specifically, quadruplex hybrid was observed for (GGG)4 in K+ buffer. Among the other modified sequences, (GIG)4, (IGI)4 and (GII)4 show parallel features, while (IIG)4 and (III)4 show no detectable conformation in the presence of either Na+ or K+. Our studies indicate that terminal lesions (IGG)4 and (GGI)4 may induce certain unknown conformations. The folding dynamics become undetectable in the presence of more than one inosine substitution except (IGI)4 in both buffer ions. In addition, both UV melting and CD melting studies implied that in most cases the K+ cation confers more thermodynamic stability compared to Na+. Collectively, our conformational studies revealed the diverse structural polymorphisms of G4 with position dependent G-to-I mutations in different ion conditions.

Integrating Genomic and Chromosomal Data: A Cytogenetic Study of Transancistrus santarosensis (Loricariidae: Hypostominae) with Characterization of a ZZ/ZW Sex Chromosome System.

The plecos (Loricariidae) fish represent a great model for cytogenetic investigations due to their variety of karyotypes, including diploid and polyploid genomes, and different types of sex chromosomes. In this study we investigate Transancistrus santarosensis a rare loricariid endemic to Ecuador, integrating cytogenetic methods with specimens' molecular identification by mtDNA, to describe the the species karyotype. We aim to verify whether sex chromosomes are cytologically identifiable and if they are associated with the accumulation of repetitive sequences present in other species of the family. The analysis of the karyotype (2n = 54 chromosomes) excludes recent centric fusion and pericentromeric inversion and suggests the presence of a ZZ/ZW sex chromosome system at an early stage of differentiation: the W chromosome is degenerated but is not characterized by the presence of differential sex-specific repetitive DNAs. Data indicate that although T. santarosensis has retained the ancestral diploid number of Loricariidae, it accumulated heterochromatin and shows non-syntenic ribosomal genes localization, chromosomal traits considered apomorphic in the family.

The Telomeric Repeats of HHV-6A Do Not Determine the Chromosome into Which the Virus Is Integrated.

Human herpes virus 6A (HHV-6A) is able to integrate into the telomeric and subtelomeric regions of human chromosomes representing chromosomally integrated HHV-6A (ciHHV-6A). The integration starts from the right direct repeat (DRR) region. It has been shown experimentally that perfect telomeric repeats (pTMR) in the DRR region are required for the integration, while the absence of the imperfect telomeric repeats (impTMR) only slightly reduces the frequency of HHV-6 integration cases. The aim of this study was to determine whether telomeric repeats within DRR may define the chromosome into which the HHV-6A integrates. We analysed 66 HHV-6A genomes obtained from public databases. Insertion and deletion patterns of DRR regions were examined. We also compared TMR within the herpes virus DRR and human chromosome sequences retrieved from the Telomere-to-Telomere consortium. Our results show that telomeric repeats in DRR in circulating and ciHHV-6A have an affinity for all human chromosomes studied and thus do not define a chromosome for integration.

Occurrence of Sex Chromosomes in Fish of the Genus Ancistrus with a New Description of Multiple Sex Chromosomes in the Ecuadorian Endemic Ancistrus clementinae (Loricariidae).

Ancistrus Kner, 1854, is the most diverse genus among the Ancistrini (Loricariidae) with 70 valid species showing a wide geographic distribution and great taxonomic and systematic complexity. To date, about 40 Ancistrus taxa have been karyotyped, all from Brazil and Argentina, but the statistic is uncertain because 30 of these reports deal with samples that have not yet been identified at the species level. This study provides the first cytogenetic description of the bristlenose catfish, Ancistrus clementinae Rendahl, 1937, a species endemic to Ecuador, aiming to verify whether a sex chromosome system is identifiable in the species and, if so, which, and if its differentiation is associated with the presence of repetitive sequences reported for other species of the family. We associated the karyotype analysis with the COI molecular identification of the specimens. Karyotype analysis suggested the presence of a ♂ZZ/♀ZW1W2 sex chromosome system, never detected before in Ancistrus, with both W1W2 chromosomes enriched with heterochromatic blocks and 18S rDNA, in addition to GC-rich repeats (W2). No differences were observed between males and females in the distribution of 5S rDNA or telomeric repeats. Cytogenetic data here obtained confirm the huge karyotype diversity of Ancistrus, both in chromosome number and sex-determination systems.

Interstitial Telomeric-like Repeats (ITR) in Seed Plants as Assessed by Molecular Cytogenetic Techniques: A Review.

The discovery of telomeric repeats in interstitial regions of plant chromosomes (ITRs) through molecular cytogenetic techniques was achieved several decades ago. However, the information is scattered and has not been critically evaluated from an evolutionary perspective. Based on the analysis of currently available data, it is shown that ITRs are widespread in major evolutionary lineages sampled. However, their presence has been detected in only 45.6% of the analysed families, 26.7% of the sampled genera, and in 23.8% of the studied species. The number of ITR sites greatly varies among congeneric species and higher taxonomic units, and range from one to 72 signals. ITR signals mostly occurs as homozygous loci in most species, however, odd numbers of ITR sites reflecting a hemizygous state have been reported in both gymnosperm and angiosperm groups. Overall, the presence of ITRs appears to be poor predictors of phylogenetic and taxonomic relatedness at most hierarchical levels. The presence of ITRs and the number of sites are not significantly associated to the number of chromosomes. The longitudinal distribution of ITR sites along the chromosome arms indicates that more than half of the ITR presences are between proximal and terminal locations (49.5%), followed by proximal (29.0%) and centromeric (21.5%) arm regions. Intraspecific variation concerning ITR site number, chromosomal locations, and the differential presence on homologous chromosome pairs has been reported in unrelated groups, even at the population level. This hypervariability and dynamism may have likely been overlooked in many lineages due to the very low sample sizes often used in cytogenetic studies.

Interstitial Arabidopsis-Type Telomeric Repeats in Asteraceae.

Tandem repeats of telomeric-like motifs at intra-chromosomal regions, known as interstitial telomeric repeats (ITR), have drawn attention as potential markers of structural changes, which might convey information about evolutionary relationships if preserved through time. Building on our previous work that reported outstanding ITR polymorphisms in the genus Anacyclus, we undertook a survey across 132 Asteraceae species, focusing on the six most speciose subfamilies and considering all the ITR data published to date. The goal was to assess whether the presence, site number, and chromosomal location of ITRs convey any phylogenetic signal. We conducted fluorescent in situ hybridization (FISH) using an Arabidopsis-type telomeric sequence as a probe on karyotypes obtained from mitotic chromosomes. FISH signals of ITR sites were detected in species of subfamilies Asteroideae, Carduoideae, Cichorioideae, Gymnarhenoideae, and Mutisioideae, but not in Barnadesioideae. Although six small subfamilies have not yet been sampled, altogether, our results suggest that the dynamics of ITR formation in Asteraceae cannot accurately trace the complex karyological evolution that occurred since the early diversification of this family. Thus, ITRs do not convey a reliable signal at deep or shallow phylogenetic levels and cannot help to delimitate taxonomic categories, a conclusion that might also hold for other important families such as Fabaceae.

A Cell Culture System to Investigate Marek's Disease Virus Integration into Host Chromosomes.

Marek's disease virus (MDV) is a highly oncogenic alphaherpesvirus that causes a devastating neoplastic disease in chickens. MDV has been shown to integrate its genome into the telomeres of latently infected and tumor cells, which is crucial for efficient tumor formation. Telomeric repeat arrays present at the ends of the MDV genome facilitate this integration into host telomeres; however, the integration mechanism remains poorly understood. Until now, MDV integration could only be investigated qualitatively upon infection of chickens. To shed further light on the integration mechanism, we established a quantitative integration assay using chicken T cell lines, the target cells for MDV latency and transformation. We optimized the infection conditions and assessed the establishment of latency in these T cells. The MDV genome was efficiently maintained over time, and integration was confirmed in these cells by fluorescence in situ hybridization (FISH). To assess the role of the two distinct viral telomeric repeat arrays in the integration process, we tested various knockout mutants in our in vitro integration assay. Efficient genome maintenance and integration was thereby dependent on the presence of the telomeric repeat arrays in the virus genome. Taken together, we developed and validated a novel in vitro integration assay that will shed light on the integration mechanism of this highly oncogenic virus into host telomeres.

The Pontastacus leptodactylus (Astacidae) Repeatome Provides Insight Into Genome Evolution and Reveals Remarkable Diversity of Satellite DNA.

Pontastacus leptodactylus is a native European crayfish species found in both freshwater and brackish environments. It has commercial importance for fisheries and aquaculture industries. Up till now, most studies concerning P. leptodactylus have focused onto gaining knowledge about its phylogeny and population genetics. However, little is known about the chromosomal evolution and genome organization of this species. Therefore, we performed clustering analysis of a low coverage genomic dataset to identify and characterize repetitive DNA in the P. leptodactylus genome. In addition, the karyogram of P. leptodactylus (2n = 180) is presented here for the first time consisting of 75 metacentric, 14 submetacentric, and a submetacentric/metacentric heteromorphic chromosome pair. We determined the genome size to be at ~18.7 gigabase pairs. Repetitive DNA represents about 54.85% of the genome. Satellite DNA repeats are the most abundant type of repetitive DNA, making up to ~28% of the total amount of repetitive elements, followed by the Ty3/Gypsy retroelements (~15%). Our study established a surprisingly high diversity of satellite repeats in P. leptodactylus. The genome of P. leptodactylus is by far the most satellite-rich genome discovered to date with 258 satellite families described. Of the five mapped satellite DNA families on chromosomes, PlSAT3-411 co-localizes with the AT-rich DAPI positive probable (peri)centromeric heterochromatin on all chromosomes, while PlSAT14-79 co-localizes with the AT-rich DAPI positive (peri)centromeric heterochromatin on one chromosome and is also located subterminally and intercalary on some chromosomes. PlSAT1-21 is located intercalary in the vicinity of the (peri)centromeric heterochromatin on some chromosomes, while PlSAT6-70 and PlSAT7-134 are located intercalary on some P. leptodactylus chromosomes. The FISH results reveal amplification of interstitial telomeric repeats (ITRs) in P. leptodactylus. The prevalence of repetitive elements, especially the satellite DNA repeats, may have provided a driving force for the evolution of the P. leptodactylus genome.

Cytogenetic analysis of Bienertia sinuspersici Akhani as the first step in genome sequencing.

BACKGROUND: C4 plants are efficient in suppressing photorespiration and enhancing carbon gain as compared to C3 plants. Bienertia sinuspersici Akhani is one of the few species in the family Amaranthaceae that can perform C4 photosynthesis within individual chlorenchyma cells, without the conventional Kranz anatomy in its leaf. This plant is salt-tolerant and is well-adapted to thrive in hot and humid climates. To date, there have been no reported cytogenetic analyses yet on this species. OBJECTIVE: This study aims to provide a cytogenetic analysis of B. sinuspersici as the first step in genome sequencing. METHODS: Fluorescence in situ hybridization (FISH) karyotype analysis was conducted using the metaphase chromosomes of B. sinuspersici probed with 5S and 45S rDNA and Arabidopsis-type telomeric repeats. RESULTS: Results of the cytogenetic analysis confirmed that B. sinuspersici has 2n = 2x = 18 consisting of nine pairs of metacentric chromosomes. Two loci of 45S rDNA were found on the distal regions of the short arm of chromosome 7. Nine loci of 5S rDNA were found in the pericentromeric regions of chromosomes 1, 3, 4, 6, and 8, which also colocalized with Arabidopsis-type telomeric repeats; while four loci in the interstitial regions of chromosome 5 and 8 can be observed. The single locus of 5S rDNA that was found in chromosome 8 appears to be hemizygous. CONCLUSION: The FISH karyotype analysis, based on the combination of rDNAs, telomeric tandem repeat markers and C0t DNA chromosome landmarks, allowed efficient chromosome identification and provided useful information in characterizing the genome of B. sinuspersici.