Showing papers on "Karyotype published in 2018"

Karyotype engineering by chromosome fusion leads to reproductive isolation in yeast

Abstract: Extant species have wildly different numbers of chromosomes, even among taxa with relatively similar genome sizes (for example, insects)1,2. This is likely to reflect accidents of genome history, such as telomere–telomere fusions and genome duplication events3–5. Humans have 23 pairs of chromosomes, whereas other apes have 24. One human chromosome is a fusion product of the ancestral state6. This raises the question: how well can species tolerate a change in chromosome numbers without substantial changes to genome content? Many tools are used in chromosome engineering in Saccharomyces cerevisiae7–10, but CRISPR–Cas9-mediated genome editing facilitates the most aggressive engineering strategies. Here we successfully fused yeast chromosomes using CRISPR–Cas9, generating a near-isogenic series of strains with progressively fewer chromosomes ranging from sixteen to two. A strain carrying only two chromosomes of about six megabases each exhibited modest transcriptomic changes and grew without major defects. When we crossed a sixteen-chromosome strain with strains with fewer chromosomes, we noted two trends. As the number of chromosomes dropped below sixteen, spore viability decreased markedly, reaching less than 10% for twelve chromosomes. As the number of chromosomes decreased further, yeast sporulation was arrested: a cross between a sixteen-chromosome strain and an eight-chromosome strain showed greatly reduced full tetrad formation and less than 1% sporulation, from which no viable spores could be recovered. However, homotypic crosses between pairs of strains with eight, four or two chromosomes produced excellent sporulation and spore viability. These results indicate that eight chromosome–chromosome fusion events suffice to isolate strains reproductively. Overall, budding yeast tolerates a reduction in chromosome number unexpectedly well, providing a striking example of the robustness of genomes to change. Yeast chromosomes have been fused to produce viable strains with only two chromosomes that are reproductively isolated from the sixteen-chromosome wild type, but otherwise show high fitness in mitosis and meiosis.

Evolutionary trends in animal ribosomal DNA loci: introduction to a new online database.

Abstract: Ribosomal DNA (rDNA) loci encoding 5S and 45S (18S-5.8S-28S) rRNAs are important components of eukaryotic chromosomes. Here, we set up the animal rDNA database containing cytogenetic information about these loci in 1343 animal species (264 families) collected from 542 publications. The data are based on in situ hybridisation studies (both radioactive and fluorescent) carried out in major groups of vertebrates (fish, reptiles, amphibians, birds, and mammals) and invertebrates (mostly insects and mollusks). The database is accessible online at www.animalrdnadatabase.com . The median number of 45S and 5S sites was close to two per diploid chromosome set for both rDNAs despite large variation (1–74 for 5S and 1–54 for 45S sites). No significant correlation between the number of 5S and 45S rDNA loci was observed, suggesting that their distribution and amplification across the chromosomes follow independent evolutionary trajectories. Each group, irrespective of taxonomic classification, contained rDNA sites at any chromosome location. However, the distal and pericentromeric positions were the most prevalent (> 75% karyotypes) for 45S loci, while the position of 5S loci was more variable. We also examined potential relationships between molecular attributes of rDNA (homogenisation and expression) and cytogenetic parameters such as rDNA positions, chromosome number, and morphology.

Comparative Oligo-FISH Mapping: An Efficient and Powerful Methodology To Reveal Karyotypic and Chromosomal Evolution.

Abstract: Developing the karyotype of a eukaryotic species relies on identification of individual chromosomes, which has been a major challenge for most nonmodel plant and animal species. We developed a novel chromosome identification system by selecting and labeling oligonucleotides (oligos) located in specific regions on every chromosome. We selected a set of 54,672 oligos (45 nt) based on single copy DNA sequences in the potato genome. These oligos generated 26 distinct FISH signals that can be used as a "bar code" or "banding pattern" to uniquely label each of the 12 chromosomes from both diploid and polyploid (4× and 6×) potato species. Remarkably, the same bar code can be used to identify the 12 homeologous chromosomes among distantly related Solanum species, including tomato and eggplant. Accurate karyotypes based on individually identified chromosomes were established in six Solanum species that have diverged for >15 MY. These six species have maintained a similar karyotype; however, modifications to the FISH signal bar code led to the discovery of two reciprocal chromosomal translocations in Solanum etuberosum and S. caripense We also validated these translocations by oligo-based chromosome painting. We demonstrate that the oligo-based FISH techniques are powerful new tools for chromosome identification and karyotyping research, especially for nonmodel plant species.

Extraordinary Genome Instability and Widespread Chromosome Rearrangements During Vegetative Growth

Abstract: The haploid genome of the pathogenic fungus Zymoseptoria tritici is contained on “core” and “accessory” chromosomes. While 13 core chromosomes are found in all strains, as many as eight accessory chromosomes show presence/absence variation and rearrangements among field isolates. The factors influencing these presence/absence polymorphisms are so far unknown. We investigated chromosome stability using experimental evolution, karyotyping, and genome sequencing. We report extremely high and variable rates of accessory chromosome loss during mitotic propagation in vitro and in planta . Spontaneous chromosome loss was observed in 2 to >50% of cells during 4 weeks of incubation. Similar rates of chromosome loss in the closely related Zymoseptoria ardabiliae suggest that this extreme chromosome dynamic is a conserved phenomenon in the genus. Elevating the incubation temperature greatly increases instability of accessory and even core chromosomes, causing severe rearrangements involving telomere fusion and chromosome breakage. Chromosome losses do not affect the fitness of Zymoseptoria tritici in vitro , but some lead to increased virulence, suggesting an adaptive role of this extraordinary chromosome instability.

MYC-containing amplicons in acute myeloid leukemia: genomic structures, evolution, and transcriptional consequences.

Abstract: Double minutes (dmin), homogeneously staining regions, and ring chromosomes are vehicles of gene amplification in cancer. The underlying mechanism leading to their formation as well as their structure and function in acute myeloid leukemia (AML) remain mysterious. We combined a range of high-resolution genomic methods to investigate the architecture and expression pattern of amplicons involving chromosome band 8q24 in 23 cases of AML (AML-amp). This revealed that different MYC-dmin architectures can coexist within the same leukemic cell population, indicating a step-wise evolution rather than a single event origin, such as through chromothripsis. This was supported also by the analysis of the chromothripsis criteria, that poorly matched the model in our samples. Furthermore, we found that dmin could evolve toward ring chromosomes stabilized by neocentromeres. Surprisingly, amplified genes (mainly PVT1) frequently participated in fusion transcripts lacking a corresponding DNA template. We also detected a significant overexpression of the circular RNA of PVT1 (circPVT1) in AML-amp cases versus AML with a normal karyotype. Our results show that 8q24 amplicons in AML are surprisingly plastic DNA structures with an unexpected association to novel fusion transcripts and circular RNAs.

Karyotype variation of CHO host cell lines over time in culture characterized by chromosome counting and chromosome painting.

Abstract: Genomic rearrangements are a common phenomenon in rapidly growing cell lines such as Chinese hamster ovary (CHO) cells, a feature that in the context of production of biologics may lead to cell line and product instability. Few methods exist to assess such genome wide instability. Here, we use the population distribution of chromosome numbers per cell as well as chromosome painting to quantify the karyotypic variation in several CHO host cell lines. CHO-S, CHO-K1 8 mM glutamine, and CHO-K1 cells adapted to grow in media containing no glutamine were analyzed over up to 6 months in culture. All three cell lines were clearly distinguishable by their chromosome number distribution and by the specific chromosome rearrangements that were present in each population. Chromosome Painting revealed a predominant karyotype for each cell line at the start of the experiment, completed by a large number of variants present in each population. Over time in culture, the predominant karyotype changed for CHO-S and CHO-K1, with the diversity increasing and new variants appearing, while CHO-K1 0 mM Gln preferred chromosome pattern increased in percent of the population over time. As control, Chinese hamster lung fibroblasts were shown to also contain an increasing number of variants over time in culture.

Tripolar chromosome segregation drives the association between maternal genotype at variants spanning PLK4 and aneuploidy in human preimplantation embryos

Abstract: Aneuploidy is prevalent in human embryos and is the leading cause of pregnancy loss. Many aneuploidies arise during oogenesis, increasing with maternal age. Superimposed on these meiotic aneuploidies are frequent errors occurring during early mitotic divisions, contributing to widespread chromosomal mosaicism. Here we reanalyzed a published dataset comprising preimplantation genetic testing for aneuploidy in 24 653 blastomere biopsies from day-3 cleavage-stage embryos, as well as 17 051 trophectoderm biopsies from day-5 blastocysts. We focused on complex abnormalities that affected multiple chromosomes simultaneously, seeking insights into their formation. In addition to well-described patterns such as triploidy and haploidy, we identified 4.7% of blastomeres possessing characteristic hypodiploid karyotypes. We inferred this signature to have arisen from tripolar chromosome segregation in normally fertilized diploid zygotes or their descendant diploid cells. This could occur via segregation on a tripolar mitotic spindle or by rapid sequential bipolar mitoses without an intervening S-phase. Both models are consistent with time-lapse data from an intersecting set of 77 cleavage-stage embryos, which were enriched for the tripolar signature among embryos exhibiting abnormal cleavage. The tripolar signature was strongly associated with common maternal genetic variants spanning the centrosomal regulator PLK4, driving the association we previously reported with overall mitotic errors. Our findings are consistent with the known capacity of PLK4 to induce tripolar mitosis or precocious M-phase upon dysregulation. Together, our data support tripolar chromosome segregation as a key mechanism generating complex aneuploidy in cleavage-stage embryos and implicate maternal genotype at a quantitative trait locus spanning PLK4 as a factor influencing its occurrence.

The Agropyron cristatum karyotype, chromosome structure and cross-genome homoeology as revealed by fluorescence in situ hybridization with tandem repeats and wheat single-gene probes

Abstract: Fluorescence in situ hybridization with probes for 45 cDNAs and five tandem repeats revealed homoeologous relationships of Agropyron cristatum with wheat. The results will contribute to alien gene introgression in wheat improvement. Crested wheatgrass (Agropyron cristatum L. Gaertn.) is a wild relative of wheat and a promising source of novel genes for wheat improvement. To date, identification of A. cristatum chromosomes has not been possible, and its molecular karyotype has not been available. Furthermore, homoeologous relationship between the genomes of A. cristatum and wheat has not been determined. To develop chromosome-specific landmarks, A. cristatum genomic DNA was sequenced, and new tandem repeats were discovered. Their distribution on mitotic chromosomes was studied by fluorescence in situ hybridization (FISH), which revealed specific patterns for five repeats in addition to 5S and 45S ribosomal DNA and rye subtelomeric repeats pSc119.2 and pSc200. FISH with one tandem repeat together with 45S rDNA enabled identification of all A. cristatum chromosomes. To analyze the structure and cross-species homoeology of A. cristatum chromosomes with wheat, probes for 45 mapped wheat cDNAs covering all seven chromosome groups were localized by FISH. Thirty-four cDNAs hybridized to homoeologous chromosomes of A. cristatum, nine hybridized to homoeologous and non-homoeologous chromosomes, and two hybridized to unique positions on non-homoeologous chromosomes. FISH using single-gene probes revealed that the wheat-A. cristatum collinearity was distorted, and important structural rearrangements were observed for chromosomes 2P, 4P, 5P, 6P and 7P. Chromosomal inversions were found for pericentric region of 4P and whole chromosome arm 6PL. Furthermore, reciprocal translocations between 2PS and 4PL were detected. These results provide new insights into the genome evolution within Triticeae and will facilitate the use of crested wheatgrass in alien gene introgression into wheat.

Historical and Clinical Perspectives on Chromosomal Translocations.

Abstract: Chromosomal translocations, rearrangements involving the exchange of segments between chromosomes, were documented in humans in 1959. The first accurately reported clinical phenotype resulting from a translocation was that of Down syndrome. In a small percentage of Down syndrome cases, an extra 21q is provided by a Robertsonian translocation chromosome, either occurring de novo or inherited from a phenotypically normal parent with the translocation chromosome and a balanced genome of 45 chromosomes. Balanced translocations, including both Robertsonian and reciprocal translocations, are typically benign, but meiosis in germ cells with balanced translocations may result in meiotic arrest and subsequent infertility, or in unbalanced gametes, with attendant risks of miscarriage and unbalanced progeny. Most reciprocal translocations are unique. A few to several percent of translocations disrupt haploinsufficient genes or their regulatory regions and result in clinical phenotypes. Balanced translocations from patients with clinical phenotypes have been valuable in mapping disease genes and in illuminating cis-regulatory regions. Mapping of discordant mate pairs from long-insert, low-pass genome sequencing now permits efficient and cost-effective discovery and nucleotide-level resolution of rearrangement breakpoints, information that is absolutely necessary for interpreting the etiology of clinical phenotypes in patients with rearrangements. Pathogenic translocations and other balanced chromosomal rearrangements constitute a class of typically highly penetrant mutation that is cryptic to both clinical microarray and exome sequencing. A significant proportion of rearrangements include additional complexity that is not visible by conventional karyotype analysis. Some proportion of patients with negative findings on exome/genome sequencing and clinical microarray will be found to have etiologic balanced rearrangements only discoverable by genome sequencing with analysis pipelines optimized to recover rearrangement breakpoints.

Sex chromosome aneuploidies

Abstract: Sex chromosome aneuploidies comprise a relatively common group of chromosome disorders characterized by the loss or gain of one or more sex chromosomes. We discuss five of the better-known sex aneuploidies: Turner syndrome (XO), Klinefelter syndrome (XXY), trisomy X (XXX), XYY, and XXYY. Despite their prevalence in the general population, these disorders are underdiagnosed and the specific genetic mechanisms underlying their phenotypes are poorly understood. Although there is considerable variation between them in terms of associated functional impairment, each disorder has a characteristic physical, cognitive, and neurologic profile. The most common cause of sex chromosome aneuploidies is nondisjunction, which can occur during meiosis or during the early stages of postzygotic development. The loss or gain of genetic material can affect all daughter cells or it may be partial, leading to tissue mosaicism. In both typical and atypical sex chromosome karyotypes, there is random inactivation of all but one X chromosome. The mechanisms by which a phenotype results from sex chromosome aneuploidies are twofold: dosage imbalance arising from a small number of genes that escape inactivation, and their endocrinologic consequences.

Quantitative proteomic and phosphoproteomic comparison of human colon cancer DLD-1 cells differing in ploidy and chromosome stability.

Abstract: Although aneuploidy is poorly tolerated during embryogenesis, aneuploidy and whole chromosomal instability (CIN) are common hallmarks of cancer, raising the question of how cancer cells can thrive in spite of chromosome aberrations. Here we present a comprehensive and quantitative proteomics analysis of isogenic DLD-1 colorectal adenocarcinoma cells lines, aimed at identifying cellular responses to changes in ploidy and/or CIN. Specifically, we compared diploid (2N) and tetraploid (4N) cells with posttetraploid aneuploid (PTA) clones and engineered trisomic clones. Our study provides a comparative data set on the proteomes and phosphoproteomes of the above cell lines, comprising several thousand proteins and phosphopeptides. In comparison to the parental 2N line, we observed changes in proteins associated with stress responses and with interferon signaling. Although we did not detect a conspicuous protein signature associated with CIN, we observed many changes in phosphopeptides that relate to fundamental cellular processes, including mitotic progression and spindle function. Most importantly, we found that most changes detectable in PTA cells were already present in the 4N progenitor line. This suggests that activation of mitotic pathways through hyper-phosphorylation likely constitutes an important response to chromosomal burden. In line with this conclusion, cells with extensive chromosome gains showed differential sensitivity toward a number of inhibitors targeting cell cycle kinases, suggesting that the efficacy of anti-mitotic drugs may depend on the karyotype of cancer cells.

Genetic interactions between specific chromosome copy number alterations dictate complex aneuploidy patterns

Abstract: Cells that contain an abnormal number of chromosomes are called aneuploid. High rates of aneuploidy in cancer are correlated with an increased frequency of chromosome missegregation, termed chromosomal instability (CIN). Both high levels of aneuploidy and CIN are associated with cancers that are resistant to treatment. Although aneuploidy and CIN are typically detrimental to cell growth, they can aid in adaptation to selective pressures. Here, we induced extremely high rates of chromosome missegregation in yeast to determine how cells adapt to CIN over time. We found that adaptation to CIN occurs initially through many different individual chromosomal aneuploidies. Interestingly, the adapted yeast strains acquire complex karyotypes with specific subsets of the beneficial aneuploid chromosomes. These complex aneuploidy patterns are governed by synthetic genetic interactions between individual chromosomal abnormalities, which we refer to as chromosome copy number interactions (CCNIs). Given enough time, distinct karyotypic patterns in separate yeast populations converge on a refined complex aneuploid state. Surprisingly, some chromosomal aneuploidies that provided an advantage early on in adaptation are eventually lost due to negative CCNIs with even more beneficial aneuploid chromosome combinations. Together, our results show how cells adapt by obtaining specific complex aneuploid karyotypes in the presence of CIN.

Sex Chromosome Evolution and Genomic Divergence in the Fish Hoplias malabaricus (Characiformes, Erythrinidae).

Abstract: The Erythrinidae family (Teleostei: Characiformes) is a small Neotropical fish group with a wide distribution throughout South America, where Hoplias malabaricus corresponds to the most widespread and cytogenetically studied taxon. This species possesses significant genetic variation, as well as huge karyotype diversity among populations, as reflected by its seven major karyotype forms (i.e. karyomorphs A-G) identified up to now. Although morphological differences in their bodies are not outstanding, H. malabaricus karyomorphs are easily identified by differences in 2n, morphology and size of chromosomes, as well as by different evolutionary steps of sex chromosomes development. Here, we performed comparative genomic hybridization (CGH) to analyse both the intra- and inter-genomic status in terms of repetitive DNA divergence among all but one (E) H. malabaricus karyomorphs.Our results indicated that they have close relationships, but with evolutionary divergences among their genomes, yielding a range of non-overlapping karyomorph-specific signals. Besides, male-specific regions were uncovered on the sex chromosomes, confirming their differential evolutionary trajectories. In conclusion, the hypothesis that H. malabaricus karyomorphs are result of speciation events was strengthened.

Tracking the evolutionary pathway of sex chromosomes among fishes: characterizing the unique XX/XY 1 Y 2 system in Hoplias malabaricus (Teleostei, Characiformes)

Abstract: The Neotropical fish, Hoplias malabaricus, is one of the most cytogenetically studied fish taxon with seven distinct karyomorphs (A–G) comprising varying degrees of sex chromosome differentiation, ranging from homomorphic to highly differentiated simple and multiple sex chromosomes. Therefore, this fish offers a unique opportunity to track evolutionary mechanisms standing behind the sex chromosome evolution and differentiation. Here, we focused on a high-resolution cytogenetic characterization of the unique XX/XY1Y2 multiple sex chromosome system found in one of its karyomorphs (G). For this, we applied a suite of conventional (Giemsa-staining, C-banding) and molecular cytogenetic approaches, including fluorescence in situ hybridization FISH (with 5S and 18S rDNAs, 10 microsatellite motifs and telomeric (TTAGGG) n sequences as probes), comparative genomic hybridization (CGH), and whole chromosome painting (WCP). In addition, we performed comparative analyses with other Erythrinidae species to discover the evolutionary origin of this unique karyomorph G-specific XY1Y2 multiple sex chromosome system. WCP experiments confirmed the homology between these multiple sex chromosomes and the nascent XX/XY sex system found in the karyomorph F, but disproved a homology with those of karyomorphs A–D and other closely related species. Besides, the putative origin of such XY1Y2 system by rearrangements of several chromosome pairs from an ancestral karyotype was also highlighted. In addition, clear identification of a male-specific region on the Y1 chromosome suggested a differential pattern of repetitive sequences accumulation. The present data suggested the origin of this unique XY1Y2 sex system, revealing evidences for the high level of plasticity of sex chromosome differentiation within the Erythrinidae.

The Drosophila Dot Chromosome: Where Genes Flourish Amidst Repeats

Abstract: The F element of the Drosophila karyotype (the fourth chromosome in Drosophila melanogaster) is often referred to as the “dot chromosome” because of its appearance in a metaphase chromosome spread. This chromosome is distinct from other Drosophila autosomes in possessing both a high level of repetitious sequences (in particular, remnants of transposable elements) and a gene density similar to that found in the other chromosome arms, ∼80 genes distributed throughout its 1.3-Mb “long arm.” The dot chromosome is notorious for its lack of recombination and is often neglected as a consequence. This and other features suggest that the F element is packaged as heterochromatin throughout. F element genes have distinct characteristics (e.g., low codon bias, and larger size due both to larger introns and an increased number of exons), but exhibit expression levels comparable to genes found in euchromatin. Mapping experiments show the presence of appropriate chromatin modifications for the formation of DNaseI hypersensitive sites and transcript initiation at the 5′ ends of active genes, but, in most cases, high levels of heterochromatin proteins are observed over the body of these genes. These various features raise many interesting questions about the relationships of chromatin structures with gene and chromosome function. The apparent evolution of the F element as an autosome from an ancestral sex chromosome also raises intriguing questions. The findings argue that the F element is a unique chromosome that occupies its own space in the nucleus. Further study of the F element should provide new insights into chromosome structure and function.

Comprehensively Characterizing the Cytological Features of Saccharum spontaneum by the Development of a Complete Set of Chromosome-Specific Oligo Probes.

Abstract: Chromosome-specific identification is a powerful technique in the study of genome structure and evolution. However, there is no reliable cytogenetic marker to unambiguously identify each of the chromosomes in sugarcane (Saccharum spp., Poaceae), which has a complex genome with a high level of ploidy and heterozygosity. In this study, we developed a set of oligonucleotide (oligo)-based probes through bioinformatic design and massive synthetization. These probes produced a clear and bright single signal in each of the chromosomes and their eight homologous chromosomes in the ancient species Saccharum spontaneum (2n = 8x = 64). Thus, they can be used as reliable markers to robustly label each of the chromosomes in S. spontaneum. We then obtained the karyotype data and established a nomenclature based on chromosomal sizes for the eight chromosomes of the octoploid S. spontaneum. In addition, we also found that the 45S and 5S rDNAs demonstrated high copy number variations among different homologous chromosomes, indicating a rapid evolution of the highly repeated sequence after polyploidization. Our fluorescence in situ hybridization (FISH) assay also demonstrated that these probes could be used as cross-species markers between or within the genera of Sorghum and Saccharum. By comparing FISH analyses, we discovered that several chromosome rearrangement events occurred in S. spontaneum, which might have contributed to the basic chromosome number reduction from 10 in sorghum to 8 in sugarcane. Consistent identification of individual chromosomes makes molecular cytogenetic study possible in sugarcane and will facilitate fine chromosomal structure and karyotype evolution of the genus Saccharum.

Chromosome map of the Siamese cobra: did partial synteny of sex chromosomes in the amniote represent "a hypothetical ancestral super-sex chromosome" or random distribution?

Abstract: Unlike the chromosome constitution of most snakes (2n=36), the cobra karyotype shows a diploid chromosome number of 38 with a highly heterochromatic W chromosome and a large morphologically different chromosome 2. To investigate the process of sex chromosome differentiation and evolution between cobras, most snakes, and other amniotes, we constructed a chromosome map of the Siamese cobra (Naja kaouthia) with 43 bacterial artificial chromosomes (BACs) derived from the chicken and zebra finch libraries using the fluorescence in situ hybridization (FISH) technique, and compared it with those of the chicken, the zebra finch, and other amniotes. We produced a detailed chromosome map of the Siamese cobra genome, focusing on chromosome 2 and sex chromosomes. Synteny of the Siamese cobra chromosome 2 (NKA2) and NKAZ were highly conserved among snakes and other squamate reptiles, except for intrachromosomal rearrangements occurring in NKA2. Interestingly, twelve BACs that had partial homology with sex chromosomes of several amniotes were mapped on the heterochromatic NKAW as hybridization signals such as repeat sequences. Sequence analysis showed that most of these BACs contained high proportions of transposable elements. In addition, hybridization signals of telomeric repeat (TTAGGG)n and six microsatellite repeat motifs ((AAGG)8, (AGAT)8, (AAAC)8, (ACAG)8, (AATC)8, and (AAAAT)6) were observed on NKAW, and most of these were also found on other amniote sex chromosomes. The frequent amplification of repeats might involve heterochromatinization and promote sex chromosome differentiation in the Siamese cobra W sex chromosome. Repeat sequences are also shared among amniote sex chromosomes, which supports the hypothesis of an ancestral super-sex chromosome with overlaps of partial syntenies. Alternatively, amplification of microsatellite repeat motifs could have occurred independently in each lineage, representing convergent sex chromosomal differentiation among amniote sex chromosomes.

Chromosome Evolution in Marsupials.

Abstract: Marsupials typically possess very large, distinctive chromosomes that make them excellent subjects for cytogenetic analysis, and the high level of conservation makes it relatively easy to track chromosome evolution. There are two speciose marsupial families with contrasting rates of karyotypic evolution that could provide insight into the mechanisms driving genome reshuffling and speciation. The family Dasyuridae displays exceptional karyotype conservation with all karyotyped species possessing a 2n = 14 karyotype similar to that predicted for the ancestral marsupial. In contrast, the family Macropodidae has experienced a higher rate of genomic rearrangement and one genus of macropods, the rock-wallabies (Petrogale), has experienced extensive reshuffling. For at least some recently diverged Petrogale species, there is still gene flow despite hybrid fertility issues, making this species group an exceptional model for studying speciation. This review highlights the unique chromosome features of marsupial chromosomes, particularly for these two contrasting families, and the value that a combined cytogenetics, genomics, and epigenomics approach will have for testing models of genome evolution and speciation.

Clonal chromosomal and genomic instability during human multipotent mesenchymal stromal cells long-term culture.

Abstract: Background aims Spontaneous mutagenesis often leads to appearance of genetic changes in cells. Although human multipotent mesenchymal stromal cells (hMSC) are considered as genetically stable, there is a risk of genomic and structural chromosome instability and, therefore, side effects of cell therapy associated with long-term effects. In this study, the karyotype, genetic variability and clone formation analyses have been carried out in the long-term culture MSC from human gingival mucosa. Methods The immunophenotype of MSC has been examined using flow cytofluorometry and short tandem repeat (STR) analysis has been carried out for authentication. The karyotype has been examined using GTG staining and mFISH, while the assessment of the aneuploidy 8 frequency has been performed using centromere specific chromosome FISH probes in interphase cells. Results The immunophenotype and STR loci combination did not change during the process of cultivation. From passage 23 the proliferative activity of cultured MSCs was significantly reduced. From passage 12 of cultivation, clones of cells with stable chromosome aberrations have been identified and the biggest of these (12%) are tetrasomy of chromosome 8. The random genetic and structural chromosomal aberrations and the spontaneous level of chromosomal aberrations in the hMSC long-term cultures were also described. Conclusions The spectrum of spontaneous chromosomal aberrations in MSC long-term cultivation has been described. Clonal chromosomal aberrations have been identified. A clone of cells with tetrasomy 8 has been detected in passage 12 and has reached the maximum size by passage 18 before and decreased along with the reduction of proliferative activity of cell line by passage 26. At later passages, the MSC line exhibited a set of cells with structural variants of the karyotype with a preponderance of normal diploid cells. The results of our study strongly suggest a need for rigorous genetic analyses of the clone formation in cultured MSCs before use in medicine.

More sex chromosomes than autosomes in the Amazonian frog Leptodactylus pentadactylus.

Abstract: Heteromorphic sex chromosomes are common in eukaryotes and largely ubiquitous in birds and mammals. The largest number of multiple sex chromosomes in vertebrates known today is found in the monotreme platypus (Ornithorhynchus anatinus, 2n = 52) which exhibits precisely 10 sex chromosomes. Interestingly, fish, amphibians, and reptiles have sex determination mechanisms that do or do not involve morphologically differentiated sex chromosomes. Relatively few amphibian species carry heteromorphic sex chromosomes, and when present, they are frequently represented by only one pair, either XX:XY or ZZ:ZW types. Here, in contrast, with several evidences, from classical and molecular cytogenetic analyses, we found 12 sex chromosomes in a Brazilian population of the smoky jungle frog, designated as Leptodactylus pentadactylus Laurenti, 1768 (Leptodactylinae), which has a karyotype with 2n = 22 chromosomes. Males exhibited an astonishing stable ring-shaped meiotic chain composed of six X and six Y chromosomes. The number of sex chromosomes is larger than the number of autosomes found, and these data represent the largest number of multiple sex chromosomes ever found among vertebrate species. Additionally, sequence and karyotype variation data suggest that this species may represent a complex of species, in which the chromosomal rearrangements may possibly have played an important role in the evolution process.

Molecular phylogenetic reconstruction and localization of the (TTAGG)n telomeric repeats in the chromosomes of Acromyrmex striatus (Roger, 1863) suggests a lower ancestral karyotype for leafcutter ants (Hymenoptera).

Abstract: Chromosome counts and karyotype characterization have proved to be important features of a genome. Chromosome changes during the diversification of ants might play an important role, given the diversity and success of Formicidae. Comparative karyotype analyses on ants have enriched and helped ant systematics. Among leafcutter ants, two major chromosome counts have been described, one frequent in Atta Fabricius, 1804 (2n = 22 in all Atta spp. whose karyotype is known) and the other frequent in Acromyrmex Mayr, 1865 (2n = 38 in the majority of species whose karyotype is known). The main exception is Acromyrmex striatus (Roger, 1863), which harbors a diploid chromosome set of 22. Here we describe the use of fluorescence in situ hybridization (FISH) with telomeric probes with (TTAGG)6 repeats to describe the telomere composition of A. striatus and to recover potential interstitial non-telomeric signals that may reflect fusion events during the evolution of leafcutter lineage from 38 to 22 chromosomes. Further, we reconstruct the ancestral chromosome numbers of the leafcutter clade based on a recently proposed molecular phylogenetic hypothesis and phylogenomic tree. Distinct signals have been observed in both extremities on the telomere chromosomes of A. striatus. Non-telomeric signals have not been retrieved in our analysis. It could be supposed that the low-numbered karyotype indeed represents the ancestral chromosome number of leafcutters. The phylogenetic reconstruction also recovered a low chromosome number from the diverse approaches implemented, suggesting that n = 11 is the most likely ancestral karyotype of the leafcutter ants and is a plesiomorphic feature shared between A. striatus and Atta spp.

Sequencing of Supernumerary Chromosomes of Red Fox and Raccoon Dog Confirms a Non-Random Gene Acquisition by B Chromosomes.

Abstract: B chromosomes (Bs) represent a variable addition to the main karyotype in some lineages of animals and plants. Bs accumulate through non-Mendelian inheritance and become widespread in populations. Despite the presence of multiple genes, most Bs lack specific phenotypic effects, although their influence on host genome epigenetic status and gene expression are recorded. Previously, using sequencing of isolated Bs of ruminants and rodents, we demonstrated that Bs originate as segmental duplications of specific genomic regions, and subsequently experience pseudogenization and repeat accumulation. Here, we used a similar approach to characterize Bs of the red fox (Vulpes vulpes L.) and the Chinese raccoon dog (Nyctereutes procyonoides procyonoides Gray). We confirm the previous findings of the KIT gene on Bs of both species, but demostrate an independent origin of Bs in these species, with two reused regions. Comparison of gene ensembles in Bs of canids, ruminants, and rodents once again indicates enrichment with cell-cycle genes, development-related genes, and genes functioning in the neuron synapse. The presence of B-chromosomal copies of genes involved in cell-cycle regulation and tissue differentiation may indicate importance of these genes for B chromosome establishment.

Sex Chromosomes of the Iconic Moth Abraxas grossulariata (Lepidoptera, Geometridae) and Its Congener A. sylvata.

Abstract: The magpie moth, Abraxas grossulariata, is an iconic species in which female heterogamety was discovered at the beginning of the 20th century. However, the sex chromosomes of this species have not yet been cytologically identified. We describe the sex chromosomes of A. grossulariata and its congener, A. sylvata. Although these species split only around 9.5 million years ago, and both species have the expected WZ/ZZ chromosomal system of sex determination and their sex chromosomes share the major ribosomal DNA (rDNA) representing the nucleolar organizer region (NOR), we found major differences between their karyotypes, including between their sex chromosomes. The species differ in chromosome number, which is 2n = 56 in A. grossularita and 2n = 58 in A. sylvata. In addition, A. grossularita autosomes exhibit massive autosomal blocks of heterochromatin, which is a very rare phenomenon in Lepidoptera, whereas the autosomes of A. sylvata are completely devoid of distinct heterochromatin. Their W chromosomes differ greatly. Although they are largely composed of female-specific DNA sequences, as shown by comparative genomic hybridization, cross-species W-chromosome painting revealed considerable sequence differences between them. The results suggest a relatively rapid molecular divergence of Abraxas W chromosomes by the independent spreading of female-specific repetitive sequences.

Hybridization-facilitated genome merger and repeated chromosome fusion after 8 million years

Abstract: The small genus Ricotia (nine species, Brassicaceae) is confined to the eastern Mediterranean. By comparative chromosome painting and a dated multi-gene chloroplast phylogeny, we reconstructed the origin and subsequent evolution of Ricotia. The ancestral Ricotia genome originated through hybridization between two older genomes with n = 7 and n = 8 chromosomes, respectively, on the Turkish mainland during the Early Miocene (c. 17.8 million years ago, Ma). Since then, the allotetraploid (n = 15) genome has been altered by two independent descending dysploidies (DD) to n = 14 in Ricotia aucheri and the Tenuifolia clade (2 spp.). By the Late Miocene (c. 10 Ma), the latter clade started to evolve in the most diverse Ricotia core clade (6 spp.), the process preceded by a DD event to n = 13. It is noteworthy that this dysploidy was mediated by a unique chromosomal rearrangement, merging together the same two chromosomes as were merged during the origin of a fusion chromosome within the paternal n = 7 genome c. 20 Ma. This shows that within a time period of c. 8 Myr genome evolution can repeat itself and that structurally very similar chromosomes may originate repeatedly from the same ancestral chromosomes by different pathways (end-to-end translocation versus nested chromosome insertion).

FISH-Based Markers Enable Identification of Chromosomes Derived From Tetraploid Thinopyrum elongatum in Hybrid Lines.

Abstract: Tetraploid Thinopyrum elongatum, which has superior abiotic stress tolerance characteristics, and exhibits resistance to stripe rust, powdery mildew, and Fusarium head blight, is a wild relative of wheat and a promising source of novel genes for wheat improvement. Currently, a high-resolution Fluorescence in situ hybridization (FISH) karyotype of tetraploid Th. elongatum is not available. To develop chromosome-specific FISH-based markers, the hexaploid Trititrigia 8801 and two accessions of tetraploid Th. elongatum were characterized by different repetitive sequences probes. We found that all E-genome chromosomes could be unambiguously identified using a combination of pSc119.2, pTa535, pTa71, and pTa713 repeats, and the E-genome chromosomes of the wild accessions and the partial amphiploid failed to exhibit any significant variation in the probe hybridization patterns. To verify the validation of these markers, the chromosome constitution of eight wheat- Th. elongatum hybrid derivatives were analyzed. We revealed that these probes could quickly detect wheat and tetraploid Th. elongatum chromosomes in hybrid lines. K16-712-1-2 was a 1E (1D) chromosome substitution line, K16-681-4 was a 2E disomic chromosome addition line, K16-562-3 was a 3E, 4E (3D, 4D) chromosome substitution line, K15-1033-8-2 contained one 4E, two 5E, and one 4ES⋅1DL Robertsonian translocation chromosome, and four other lines carried monosomic 4E, 5E, 6E, and 7E chromosome, respectively. Furthermore, the E-genome specific molecular markers analysis corresponded perfectly with the FISH results. The developed FISH markers will facilitate rapid identification of tetraploid Th. elongatum chromosomes in wheat improvement programs and allow appropriate alien chromosome transfer.

Extensive karyotype variability of African fish genus Nothobranchius (Cyprinodontiformes)

Abstract: Karyotypes of 65 species of the genus Nothobranchius Peters, 1868 were reviewed and of those 35 examined first time The results of present study have shown that fishes of the genus Nothobranchius possessed highly diverse karyotypes The diploid chromosome number (2n) ranged from 16 to 50 The most frequent 2n was 2n = 36 (in 35 species) while the second one 2n = 38 (in 13 species) Proportion of biarmed chromosomes varied from 0 to 95% between species Diploid chromosome number variability apparently exists as a result of chromosomal fusions or fissions and extensive karyotypic formula alterations promoting by inversions Multiple sex chromosomes of system X1X1X2X2/X1X2Y type were found only in karyotypes of 5 species The extensive karyotype variability, unusual for teleosts, of genus Nothobranchius can be likely associated with the characteristics of its life cycle and inhabiting under unstable environment of East African savannah temporal pools

Preliminary analysis of numerical chromosome abnormalities in reciprocal and Robertsonian translocation preimplantation genetic diagnosis cases with 24-chromosomal analysis with an aCGH/SNP microarray

Abstract: The aim of this study was to determine whether an interchromosomal effect (ICE) occurred in embryos obtained from reciprocal translocation (rcp) and Robertsonian translocation (RT) carriers who were following a preimplantation genetic diagnosis (PGD) with whole chromosome screening with an aCGH and SNP microarray. We also analyzed the chromosomal numerical abnormalities in embryos with aneuploidy in parental chromosomes that were not involved with a translocation and balanced in involved parental translocation chromosomes. This retrospective study included 832 embryos obtained from rcp carriers and 382 embryos from RT carriers that were biopsied in 139 PGD cycles. The control group involved embryos obtained from age-matched patient karyotypes who were undergoing preimplantation genetic screening (PGS) with non-translocation, and 579 embryos were analyzed in the control group. A single blastomere at the cleavage stage or trophectoderm from a blastocyst was biopsied, and 24-chromosomal analysis with an aCGH/SNP microarray was conducted using the PGD/PGS protocols. Statistical analyses were implemented on the incidences of cumulative aneuploidy rates between the translocation carriers and the control group. Reliable results were obtained from 138 couples, among whom only one patient was a balanced rcp or RT translocation carrier, undergoing PGD testing in our center from January 2012 to June 2014. For day 3 embryos, the aneuploidy rates were 50.7% for rcp carriers and 49.1% for RT carriers, compared with the control group, with 44.8% at a maternal age < 36 years. When the maternal age was ≥ 36 years, the aneuploidy rates were increased to 61.1% for rcp carriers, 56.7% for RT carriers, and 60.3% for the control group. There were no significant differences. In day 5 embryos, the aneuploidy rates were 24.5% for rcp carriers and 34.9% for RT carriers, compared with the control group with 53.6% at a maternal age < 36 years. When the maternal age was ≥ 36 years, the aneuploidy rates were 10.7% for rcp carriers, 26.3% for RT carriers, and 57.1% for the control group. The cumulative aneuploidy rates of chromosome translocation carriers were significantly lower than the control group. No ICE was observed in cleavage and blastocyst stage embryos obtained from these carriers. Additionally, the risk of chromosomal numerical abnormalities was observed in each of the 23 pairs of autosomes or sex chromosomes from day 3 and day 5 embryos. There was not enough evidence to prove that ICE was present in embryos derived from both rcp and RT translocation carriers, regardless of the maternal age. However, chromosomal numerical abnormalities were noticed in 23 pairs of autosomes and sex chromosomes in parental structurally normal chromosomes. Thus, 24-chromosomal analysis with an aCGH/SNP microarray PGD protocol is required to decrease the risks of failure to diagnose aneuploidy in structurally normal chromosomes.

A Markov chain for numerical chromosomal instability in clonally expanding populations.

Abstract: Cancer cells frequently undergo chromosome missegregation events during mitosis, whereby the copies of a given chromosome are not distributed evenly among the two daughter cells, thus creating cells with heterogeneous karyotypes. A stochastic model tracing cellular karyotypes derived from clonal populations over hundreds of generations was recently developed and experimentally validated, and it was capable of predicting favorable karyotypes frequently observed in cancer. Here, we construct and study a Markov chain that precisely describes karyotypic evolution during clonally expanding cancer cell populations. The Markov chain allows us to directly predict the distribution of karyotypes and the expected size of the tumor after many cell divisions without resorting to computationally expensive simulations. We determine the limiting karyotype distribution of an evolving tumor population, and quantify its dependency on several key parameters including the initial karyotype of the founder cell, the rate of whole chromosome missegregation, and chromosome-specific cell viability. Using this model, we confirm the existence of an optimal rate of chromosome missegregation probabilities that maximizes karyotypic heterogeneity, while minimizing the occurrence of nullisomy. Interestingly, karyotypic heterogeneity is significantly more dependent on chromosome missegregation probabilities rather than the number of cell divisions, so that maximal heterogeneity can be reached rapidly (within a few hundred generations of cell division) at chromosome missegregation rates commonly observed in cancer cell lines. Conversely, at low missegregation rates, heterogeneity is constrained even after thousands of cell division events. This leads us to conclude that chromosome copy number heterogeneity is primarily constrained by chromosome missegregation rates and the risk for nullisomy and less so by the age of the tumor. This model enables direct integration of karyotype information into existing models of tumor evolution based on somatic mutations.