Karyotypes of water frogs from the Pelophylax esculentus complex: results of cross-species chromosomal painting.

Amphibian species have the largest genome size enriched with repetitive sequences and relatively similar karyotypes. Moreover, many amphibian species frequently hybridize causing nuclear and mitochondrial genome introgressions. In addition, hybridization in some amphibian species may lead to clonality and polyploidization. All such events were found in water frogs from the genus Pelophylax. Among the species within the genus Pelophylax, P. esculentus complex is the most widely distributed and well-studied. This complex includes two parental species, P. ridibundus and P. lessonae, and their hybrids, P. esculentus, reproducing hemiclonally. Parental species and their hybrids have similar but slightly polymorphic karyotypes, so their precise identification is still required. Here, we have developed a complete set of 13 chromosome painting probes for two parental species allowing the precise identification of all chromosomes. Applying chromosomal painting, we identified homologous chromosomes in both parental species and orthologous chromosomes in their diploid hemiclonal hybrids. Comparative painting did not reveal interchromosomal exchanges between the studied water frog species and their hybrids. Using cross-specific chromosome painting, we detected unequal distribution of the signals along chromosomes suggesting the presence of species-specific tandem repeats. Application of chromosomal paints to the karyotypes of hybrids revealed differences in the intensity of staining for P. ridibundus and P. lessonae chromosomes. Thus, both parental genomes have a divergence in unique sequences. Obtained chromosome probes may serve as a powerful tool to unravel chromosomal evolution in phylogenetically related species, identify individual chromosomes in different cell types, and investigate the elimination of chromosomes in hybrid water frogs.

Variants of a major DNA satellite discriminate parental subgenomes in a hybrid parthenogenetic lizard Darevskia unisexualis (Darevsky, 1966).

Hybrid parthenogenetic animals are an exceptionally interesting model for studying the mechanisms and evolution of sexual and asexual reproduction. A diploid parthenogenetic lizard Darevskia unisexualis is a result of an ancestral cross between a maternal species Darevskia raddei nairensis and a paternal species Darevskia valentini and presents a unique opportunity for a cytogenetic and computational analysis of a hybrid karyotype. Our previous results demonstrated a significant divergence between the pericentromeric DNA sequences of the parental Darevskia species; however, an in-depth comparative study of their pericentromeres is still lacking. Here, using target sequencing of microdissected pericentromeric regions, we reveal and compare the repertoires of the pericentromeric tandem repeats of the parental Darevskia lizards. We found species-specific sequences of the major pericentromeric tandem repeat CLsat, which allowed computational prediction and experimental validation of fluorescent DNA probes discriminating parental chromosomes within the hybrid karyotype of D. unisexualis. Moreover, we have implemented a generalizable computational method, based on the optimization of the Levenshtein distance between tandem repeat monomers, for finding species-specific fluorescent probes for pericentromere staining. In total, we anticipate that our comparative analysis of Darevskia pericentromeric repeats, the species-specific fluorescent probes that we found and the pipeline that we developed will form a basis for the future detailed cytogenomic studies of a wide range of natural and laboratory hybrids.

Against the mainstream: exceptional evolutionary stability of ZW sex chromosomes across the fish families Triportheidae and Gasteropelecidae (Teleostei: Characiformes).

Teleost fishes exhibit a breath-taking diversity of sex determination and differentiation mechanisms. They encompass at least nine sex chromosome systems with often low degree of differentiation, high rate of inter- and intra-specific variability, and frequent turnovers. Nevertheless, several mainly female heterogametic systems at an advanced stage of genetic differentiation and high evolutionary stability have been also found across teleosts, especially among Neotropical characiforms. In this study, we aim to characterize the ZZ/ZW sex chromosome system in representatives of the Triportheidae family (Triportheus auritus, Agoniates halecinus, and the basal-most species Lignobrycon myersi) and its sister clade Gasteropelecidae (Carnegiella strigata, Gasteropelecus levis, and Thoracocharax stellatus). We applied both conventional and molecular cytogenetic approaches including chromosomal mapping of 5S and 18S ribosomal DNA clusters, cross-species chromosome painting (Zoo-FISH) with sex chromosome-derived probes and comparative genomic hybridization (CGH). We identified the ZW sex chromosome system for the first time in A. halecinus and G. levis and also in C. strigata formerly reported to lack sex chromosomes. We also brought evidence for possible mechanisms underlying the sex chromosome differentiation, including inversions, repetitive DNA accumulation, and exchange of genetic material. Our Zoo-FISH experiments further strongly indicated that the ZW sex chromosomes of Triportheidae and Gasteropelecidae are homeologous, suggesting their origin before the split of these lineages (approx. 40-70 million years ago). Such extent of sex chromosome stability is almost exceptional in teleosts, and hence, these lineages afford a special opportunity to scrutinize unique evolutionary forces and pressures shaping sex chromosome evolution in fishes and vertebrates in general.

Evolutionary breakpoint regions and chromosomal remodeling in Harttia (Siluriformes: Loricariidae) species diversification.

The Neotropical armored catfish genus Harttia presents a wide variation of chromosomal rearrangements among its representatives. Studies indicate that translocation and Robertsonian rearrangements have triggered the karyotype evolution in the genus, including differentiation of sex chromosome systems. However, few studies used powerful tools, such as comparative whole chromosome painting, to clarify this highly diversified scenario. Here, we isolated probes from the X1 (a 5S rDNA carrier) and the X2 (a 45S rDNA carrier) chromosomes of Harttia punctata, which displays an X1X1X2X2/X1X2Y multiple sex chromosome system. Those probes were applied in other Harttia species to evidence homeologous chromosome blocks. The resulting data reinforce that translocation events played a role in the origin of the X1X2Y sex chromosome system in H. punctata. The repositioning of homologous chromosomal blocks carrying rDNA sites among ten Harttia species has also been demonstrated. Anchored to phylogenetic data it was possible to evidence some events of the karyotype diversification of the studied species and to prove an independent origin for the two types of multiple sex chromosomes, XX/XY1Y2 and X1X1X2X2/X1X2Y, that occur in Harttia species. The results point to evolutionary breakpoint regions in the genomes within or adjacent to rDNA sites that were widely reused in Harttia chromosome remodeling.

Evolution of the parthenogenetic rock lizard hybrid karyotype: Robertsonian translocation between two maternal chromosomes in Darevskia rostombekowi.

Darevskia rostombekowi, the most outstanding of the seven known parthenogenetic species in the genus Darevskia, is the result of an ancestral cross between two bisexual species Darevskia raddei and Darevskia portschinskii. The chromosomal set of this species includes a unique submetacentric autosomal chromosome; the origin of this chromosome was unresolved as only acrocentric chromosomes are described in the karyotypes of Darevskia genus normally. Here, we applied a suite of molecular cytogenetic techniques, including the mapping of telomeric (TTAGGG) n repeats using fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and whole-chromosome painting (WCP) in both D. rostombekowi and parental (D. portschinskii and D. raddei) species. The obtained results in total suggest that a de novo chromosomal rearrangement via Robertsonian translocation (centric fusion) between two maternal (D. raddei) acrocentric chromosomes of different size was involved in the formation of this unique submetacentric chromosome present in the parthenogenetic species D. rostombekowi. Our findings provide new data in specific and rapid evolutional processes of a unisexual reptile species karyotype.

Meiotic synapsis of homeologous chromosomes and mismatch repair protein detection in the parthenogenetic rock lizard Darevskia unisexualis.

Parthenogenetic species of Caucasian rock lizards of the genus Darevksia are important evidence for reticulate evolution and speciation by hybridization in vertebrates. Female-only lineages formed through interspecific hybridization have been discovered in many groups. Nevertheless, critical mechanisms of oogenesis and specifics of meiosis that provide long-term stability of parthenogenetic species are still unknown. Here we report cytogenetic characteristics of somatic karyotypes and meiotic prophase I nuclei in the diploid parthenogenetic species Darevskia unisexualis from the new population "Keti" in Armenia which contains an odd number of chromosomes 2n = 37, instead of the usual 2n = 38. We revealed 36 acrocentric chromosomes and a single metacentric autosomal chromosome, resulting from Robertsonian translocation. Comparative genomic hybridization revealed that chromosome fusion occurred between two chromosomes inherited from the maternal species, similar to another parthenogenetic species D. rostombekowi. To trace the chromosome behaviour in meiosis, we performed an immunocytochemical study of primary oocytes' spread nuclei and studied chromosome synapsis during meiotic prophase I in D. unisexualis based on analysis of synaptonemal complexes (SCs). We found meiotic SC-trivalent composed of one metacentric and two acrocentric chromosomes. We confirmed that the SC was assembled between homeologous chromosomes inherited from two parental species. Immunostaining of the pachytene and diplotene nuclei revealed a mismatch repair protein MLH1 loaded to all autosomal SC bivalents. Possible mechanisms of meiotic recombination between homeologous chromosomes are discussed.

The association of three DNA repair genes polymorphisms on the frequency of chromosomal alterations detected by fluorescence in situ hybridization.

PURPOSE: Gas station workers (GSWs) are exposed to carcinogenic agents. The aim was to study the association of high somatic chromosome alterations (CAs) rates in the blood of GSWs and the polymorphisms of three genes playing a role in DNA double-strand break repair. METHODS: This is a cross-sectional study with 114 GSWs and 115 age-matched controls. Cytogenetic analyses, blood exams, medical interviews and genotypes for RAD51/G135C (rs1801320), ATM/P1054R (rs1800057) and CHEK2/T470C (rs17879961) genes were performed. RESULTS: The CA rate in GSWs was 9.8 CAs/1000 metaphases, and 19.1% of the workers had > 10 CAs per 1000 metaphases (group two). GSWs had decreased levels of monocytes (P = 0.024) in their blood exams. The number of variant alleles of the RAD51/G135C polymorphism was higher in GSWs (P = 0.011) compared to the controls, and were associated with enhanced number of CAs per worker (P = 0.008). No allele variant was found for CHEK2/T470C in this study. CONCLUSION: The RAD51/G135C polymorphism appears to be related to genome instability in gas station workers. Increasing the knowledge of DNA repair gene variations involved in maintaining genomic stability in GSWs may be crucial for future cancer prevention.

Heterochromatic regions in Japanese quail chromosomes: comprehensive molecular-cytogenetic characterization and 3D mapping in interphase nucleus.

Chromosomes of Japanese quail (Coturnix coturnix japonica, 2n=78), a galliform domestic species closely related to chicken, possess multiple heterochromatic segments. Due to the difficulties in careful analysis of such heterochromatic regions, there is a lack of data on their DNA composition, epigenetic status, as well as spatial distribution in interphase nucleus. In the present study, we applied giant lampbrush chromosome (LBC) microdissection for high-resolution analysis of quail centromeric regions of macrochromosomes and polymorphic short arms of submetacentric microchromosomes. FISH with the dissected material on mitotic and meiotic chromosomes indicated that in contrast to centromeres of chicken macrochromosomes, which are known to harbor chromosome-specific and, in some cases, tandem repeat-free sequences, centromeres of quail macroautosomes (CCO1-CCO11) have canonical organization. CCO1-CCO11 centromeres possess massive blocks of common DNA repeats demonstrating transcriptional activity at LBC stage. These repeats seem to have been subjected to chromosome size-correlated homogenization previously described primarily for avian microchromosomes. In addition, comparative FISH on chicken chromosomes supported the previous data on centromere repositioning events during galliform karyotype evolution. In interphase nucleus of different cell types, repetitive elements specific for microchromosome short arms constitute the material of prominent centrally located chromocenters enriched with markers of constitutive heterochromatin and rimmed with clusters of microchromosomal centromeric BglII-repeat. Thus, clustering of such repeats is responsible for the peculiar architecture of quail interphase nucleus. In contrast, centromere repeats of the largest macrochromosomes (CCO1 and CCO2) are predominantly localized in perinuclear heterochromatin. The possible involvement of the isolated repeats in radial genome organization is discussed.

Small supernumerary marker chromosomes (sSMC) and male infertility: characterization of five new cases, review of the literature, and perspectives.

PURPOSE: To characterize small supernumerary marker chromosomes (sSMC) in infertile males RESEARCH QUESTION: Are molecular cytogenetic methods still relevant for the identification and characterization of sSMC in the era of next-generation sequencing? METHODS: In this paper, we report five males with oligoasthenozoospermia or azoospermia with a history of recurrent pregnancy loss in partnership in four cases. R-banding karyotyping and fluorescence in situ hybridization (FISH) analysis were performed and showed sSMC in all five cases. Microdissection and reverse-FISH were performed in one case. RESULTS: One sSMC, each, was derived from chromosome 15 and an X-chromosome; two sSMC were derivatives of chromosome 22. The fifth sSMC was a ring chromosome 4 complemented by a deletion of the same region 4p14 to 4p16.1 in one of the normal chromosomes 4. All markers were mosaics except one of sSMC(22). CONCLUSION: Through this study, we emphasize the necessity of a proper combination of high-throughput techniques with conventional cytogenetic and FISH methods. This could provide a personalized diagnostic and accurate results for the patients suffering from infertility or RPL. We also highlight FISH analyses, which are essential tools for detecting sSMC in infertile patients. In fact, despite its entire composition of heterochromatin, sSMC can have effects on spermatogenesis by producing mechanical perturbations during meiosis and increasing meiotic nondisjunction rate. This would contribute to understand the exact chromosomal mechanism disrupting the natural and the assisted reproduction leading to offer a personalized support.

Doxorubicin-Induced Translocation of mtDNA into the Nuclear Genome of Human Lymphocytes Detected Using a Molecular-Cytogenetic Approach.

Translocation of mtDNA in the nuclear genome is an ongoing process that contributes to the development of pathological conditions in humans. However, the causal factors of this biological phenomenon in human cells are poorly studied. Here we analyzed mtDNA insertions in the nuclear genome of human lymphocytes after in vitro treatment with doxorubicin (DOX) using a fluorescence in situ hybridization (FISH) technique. The number of mtDNA insertions positively correlated with the number of DOX-induced micronuclei, suggesting that DOX-induced chromosome breaks contribute to insertion events. Analysis of the odds ratios (OR) revealed that DOX at concentrations of 0.025 and 0.035 µg/mL significantly increases the rate of mtDNA insertions (OR: 3.53 (95% CI: 1.42-8.76, p < 0.05) and 3.02 (95% CI: 1.19-7.62, p < 0.05), respectively). Analysis of the distribution of mtDNA insertions in the genome revealed that DOX-induced mtDNA insertions are more frequent in larger chromosomes, which are more prone to the damaging action of DOX. Overall, our data suggest that DOX-induced chromosome damage can be a causal factor for insertions of mtDNA in the nuclear genome of human lymphocytes. It can be assumed that the impact of a large number of external and internal mutagenic factors contributes significantly to the origin and amount of mtDNA in nuclear genomes.

Small supernumerary marker chromosomes: A legacy of trisomy rescue?

We studied by a whole genomic approach and trios genotyping, 12 de novo, nonrecurrent small supernumerary marker chromosomes (sSMC), detected as mosaics during pre- or postnatal diagnosis and associated with increased maternal age. Four sSMCs contained pericentromeric portions only, whereas eight had additional non-contiguous portions of the same chromosome, assembled together in a disordered fashion by repair-based mechanisms in a chromothriptic event. Maternal hetero/isodisomy was detected with a paternal origin of the sSMC in some cases, whereas in others two maternal alleles in the sSMC region and biparental haplotypes of the homologs were detected. In other cases, the homologs were biparental while the sSMC had the same haplotype of the maternally inherited chromosome. These findings strongly suggest that most sSMCs are the result of a multiple-step mechanism, initiated by maternal meiotic nondisjunction followed by postzygotic anaphase lagging of the supernumerary chromosome and its subsequent chromothripsis.

Tracking the evolutionary pathway of sex chromosomes among fishes: characterizing the unique XX/XY1Y2 system in Hoplias malabaricus (Teleostei, Characiformes).

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.

Chromosome Microdissection on Semi-Archived Material.

Glass needle-based chromosome microdissection (midi) is a standard approach developed in the 1980s and remains more frequently applied in testing than the comparable technique using laser-based platforms. As the amount of DNA extracted by this technique is minimal and often in the range of picograms, the isolated DNA must be further amplified prior to use; the isolated amplified product can be readily utilized in multiple molecular research and diagnostic investigation. DNA libraries created by midi are either chromosome- or chromosome-region-specific. However, a critical component to this process is the need for timely chromosome preparation via the air-drying method not to exceed a ~2-3 h before midi is performed. Failure of this time-sensitive step often results in the chromosomes drying out after dropping, and upon initiation of the midi technique, the dissected material can jump away while touching by the needle, and collection of a suitable sample is inhibited. Herein, we demonstrate with a simple adaptation of the standard procedure, midi can be performed on semi-archived material stored for longer periods at -20°C. Thus, the critical step to obtain well-spread chromosome preparations can be completed under established conditions, for example, in the primary laboratory, stored at -20°C, and sent directly to specialized reference laboratories offering midi. In our study, we were able to obtain high-quality DNA libraries, as verified by gel electrophoreses and reverse fluorescence in situ hybridization, via midi extracted chromosome spreads derived from human, fish, snake, lampbrush, and insect stored for up to 6 months. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

Deciphering the Origin and Evolution of the X1X2Y System in Two Closely-Related Oplegnathus Species (Oplegnathidae and Centrarchiformes).

Oplegnathus fasciatus and O. punctatus (Teleostei: Centrarchiformes: Oplegnathidae), are commercially important rocky reef fishes, endemic to East Asia. Both species present an X1X2Y sex chromosome system. Here, we investigated the evolutionary forces behind the origin and differentiation of these sex chromosomes, with the aim to elucidate whether they had a single or convergent origin. To achieve this, conventional and molecular cytogenetic protocols, involving the mapping of repetitive DNA markers, comparative genomic hybridization (CGH), and whole chromosome painting (WCP) were applied. Both species presented similar 2n, karyotype structure and hybridization patterns of repetitive DNA classes. 5S rDNA loci, besides being placed on the autosomal pair 22, resided in the terminal region of the long arms of both X1 chromosomes in females, and on the X1 and Y chromosomes in males. Furthermore, WCP experiments with a probe derived from the Y chromosome of O. fasciatus (OFAS-Y) entirely painted the X1 and X2 chromosomes in females and the X1, X2, and Y chromosomes in males of both species. CGH failed to reveal any sign of sequence differentiation on the Y chromosome in both species, thereby suggesting the shared early stage of neo-Y chromosome differentiation. Altogether, the present findings confirmed the origin of the X1X2Y sex chromosomes via Y-autosome centric fusion and strongly suggested their common origin.

Chromosomal Evolution and Evolutionary Relationships of Lebiasina Species (Characiformes, Lebiasinidae).

We present the first cytogenetic data for Lebiasina bimaculata and L. melanoguttata with the aim of (1) investigating evolutionary events within Lebiasina and their relationships with other Lebiasinidae genera and (2) checking the evolutionary relationships between Lebiasinidae and Ctenoluciidae. Both species have a diploid number 2n = 36 with similar karyotypes and microsatellite distribution patterns but present contrasting C-positive heterochromatin and CMA3+ banding patterns. The remarkable interstitial series of C-positive heterochromatin occurring in L. melanoguttata is absent in L. bimaculata. Accordingly, L. bimaculata shows the ribosomal DNA sites as the only GC-rich (CMA3+) regions, while L. melanoguttata shows evidence of a clear intercalated CMA3+ banding pattern. In addition, the multiple 5S and 18S rDNA sites in L. melanogutatta contrast with single sites present in L. bimaculata. Comparative genomic hybridization (CGH) experiments also revealed a high level of genomic differentiation between both species. A polymorphic state of a conspicuous C-positive, CMA3+, and (CGG)n band was found only to occur in L. bimaculata females, and its possible relationship with a nascent sex chromosome system is discussed. Whole chromosome painting (WCP) and CGH experiments indicate that the Lebiasina species examined and Boulengerella maculata share similar chromosomal sequences, thus supporting the relatedness between them and the evolutionary relationships between the Lebiasinidae and Ctenoluciidae families.

Deciphering the Evolutionary History of Arowana Fishes (Teleostei, Osteoglossiformes, Osteoglossidae): Insight from Comparative Cytogenomics.

Arowanas (Osteoglossinae) are charismatic freshwater fishes with six species and two genera (Osteoglossum and Scleropages) distributed in South America, Asia, and Australia. In an attempt to provide a better assessment of the processes shaping their evolution, we employed a set of cytogenetic and genomic approaches, including i) molecular cytogenetic analyses using C- and CMA3/DAPI staining, repetitive DNA mapping, comparative genomic hybridization (CGH), and Zoo-FISH, along with ii) the genotypic analyses of single nucleotide polymorphisms (SNPs) generated by diversity array technology sequencing (DArTseq). We observed diploid chromosome numbers of 2n = 56 and 54 in O. bicirrhosum and O. ferreirai, respectively, and 2n = 50 in S. formosus, while S. jardinii and S. leichardti presented 2n = 48 and 44, respectively. A time-calibrated phylogenetic tree revealed that Osteoglossum and Scleropages divergence occurred approximately 50 million years ago (MYA), at the time of the final separation of Australia and South America (with Antarctica). Asian S. formosus and Australian Scleropages diverged about 35.5 MYA, substantially after the latest terrestrial connection between Australia and Southeast Asia through the Indian plate movement. Our combined data provided a comprehensive perspective of the cytogenomic diversity and evolution of arowana species on a timescale.

Evidence for a Robertsonian fusion in Solea senegalensis (Kaup, 1858) revealed by zoo-FISH and comparative genome analysis.

BACKGROUND: Solea senegalensis (Kaup, 1858) is a commercially important flatfish species, belonging to the Pleuronectiformes order. The taxonomy of this group has long been controversial, and the karyotype of the order presents a high degree of variability in diploid number, derived from chromosomal rearrangements such as Robertsonian fusions. Previously it has been proposed that the large metacentric chromosome of S. senegalensis arises from this kind of chromosome rearrangement and that this is a proto-sex chromosome. RESULTS: In this work, the Robertsonian origin of the large metacentric chromosome of S. senegalensis has been tested by the Zoo-FISH technique applied to two species of the Soleidae family (Dicologlossa cuneata and Dagetichthys lusitanica), and by comparative genome analysis with Cynoglossus semilaevis. From the karyotypic analysis we were able to determine a chromosome complement comprising 2n = 50 (FN = 54) in D. cuneata and 2n = 42 (FN = 50) in D. lusitanica. The large metacentric painting probe gave consistent signals in four acrocentric chromosomes of the two Soleidae species; and the genome analysis proved a common origin with four chromosome pairs of C. semilaevis. As a result of the genomic analysis, up to 61 genes were annotated within the thirteen Bacterial Artificial Chromosome clones analysed. CONCLUSIONS: These results confirm that the large metacentric chromosome of S. senegalensis originated from a Robertsonian fusion and provide new data about the chromosome evolution of S. senegalensis in particular, and of Pleuronectiformes in general.

Small Supernumerary Marker Chromosome May Provide Information on Dosage-insensitive Pericentric Regions in Human.

BACKGROUND: Cytogenetically visible chromosomal imbalances in humans are deleterious and adverse in the majority of the cases. However, healthy persons living with chromosomal imbalances in the range of several megabasepairs (Mbps) in size, like carriers of small Supernumerary Marker Chromosomes (sSMCs) exist. MATERIALS & METHODS: The identification of healthy sSMC carriers with euchromatic centromere-near (ECN) imbalances led to the following proposal: ECN-regions do not contain any dosage sensitive genes. Due to own previous work, dosage-insensitive pericentric ECN-regions were already determined with an accuracy of 0.3 and 5 Mbp. Based on this data we established 43 new pericentromeric probe sets spanning about 3-5 Mbp of each euchromatic human chromosome arm starting from the known insensitive regions towards distal. Such so called pericentromeric-critical region fluorescence in situ hybridization (PeCR-FISH) probe sets were applied exemplarily and successful here in 15 sSMC cases as available from the Else Kröner-Fresenius-sSMC-cellbank . CONCLUSION: Most of the involved sSMC breakpoints could be characterized as a higher resolution than before. An unexpected result was that in 5/15 cases cryptic mosaicism was characterized. The latter is also to be considered to have potentially an influence on the clinical outcome in these so-called discontinuous sSMCs. Overall, the suitability of PeCR-FISH to characterize sSMCs was proven; the potential of this probe set to further delineate sizes of dosage insensitive pericentric regions is obvious but dependent on suited cases. Furthermore, discontinuous sSMCs can be identified by this approach and this new subtype of sSMC needs to be studied in more detail in future.

Early Stages of XY Sex Chromosomes Differentiation in the Fish Hoplias malabaricus (Characiformes, Erythrinidae) Revealed by DNA Repeats Accumulation.

BACKGROUND: Species with 'young' or nascent sex chromosomes provide unique opportunities to understand early evolutionary mechanisms (e.g. accumulation of repetitive sequences, cessation of recombination and gene loss) that drive the evolution of sex chromosomes. Among vertebrates, fishes exhibit highly diverse and a wide spectrum of sex-determining mechanisms and sex chromosomes, ranging from cryptic to highly differentiated ones, as well as, from simple to multiple sex chromosome systems. Such variability in sex chromosome morphology and composition not only exists within closely related taxa, but often within races/populations of the same species. Inside this context, the wolf fish Hoplias malabaricus offers opportunity to investigate the evolution of morphologically variable sex chromosomes within a species complex, as homomorphic to highly differentiated sex chromosome systems occur among its different karyomorphs. MATERIALS & METHODS: To discover various evolutionary stages of sex chromosomes and to compare their sequence composition among the wolf fish´s karyomorphs, we applied multipronged molecular cytogenetic approaches, including C-banding, repetitive DNAs mapping, Comparative Genomic Hybridization (CGH) and Whole Chromosomal Painting (WCP). Our study was able to characterize a cryptically differentiated XX/XY sex chromosome system in the karyomorph F of this species. CONCLUSION: The Y chromosome was clearly identified by an interstitial heterochromatic block on the short arms, primarily composed of microsatellite motifs and retrotransposons. Additionally, CGH also identified a male specific chromosome region in the same chromosomal location, implying that the accumulation of these repeats may have initiated the Y chromosome differentiation, as well as played a critical role towards the evolution and differentiation of sex chromosomes in various karyomorphs of this species.