Cytogenetic Approaches Research Articles

Novel centromeric repetitive DNA elements reveal karyotype dynamics in polyploid sainfoin (Onobrychis viciifolia).

Polyploidy is a common feature in eukaryotes with one of paramount consequences leading to better environmental adaptation. Heterochromatin is often located at telomeres and centromeres and contains repetitive DNA sequences. Sainfoin (Onobrychis viciifolia) is an important perennial forage legume for sustainable agriculture. However, there are only a few studies on the sainfoin genome and chromosomes. In this study, novel tandem repetitive DNA sequences of the sainfoin genome (OnVi180, OnVi169, OnVi176 and OnVidimer) were characterized using bioinformatics, molecular and cytogenetic approaches. The OnVi180 and OnVi169 elements colocalized within functional centromeres. The OnVi176 and OnVidimer elements were localized in centromeric, subtelomeric and interstitial regions. We constructed a sainfoin karyotype that distinguishes the seven basic chromosome groups. Our study provides the first detailed description of heterochromatin and chromosome structure of sainfoin and proposes an origin of heterozygous ancestral genomes, possibly from the same ancestral diploid species, not necessarily from different species, or for chromosome rearrangements after polyploidy. Overall, we discuss our novel and complementary findings in a polyploid crop with unique and complex chromosomal features.

Satellitome analysis on the pale-breasted thrush Turdus leucomelas (Passeriformes; Turdidae) uncovers the putative co-evolution of sex chromosomes and satellite DNAs

Do all birds' sex chromosomes follow the same canonical one-way direction of evolution? We combined cytogenetic and genomic approaches to analyze the process of the W chromosomal differentiation in two selected Passeriform species, named the Pale-breasted Thrush Turdus leucomelas and the Rufous-bellied thrush T. rufiventris. We characterized the full catalog of satellite DNAs (satellitome) of T. leucomelas, and the 10 TleSatDNA classes obtained together with 16 microsatellite motifs were in situ mapped in both species. Additionally, using Comparative Genomic Hybridization (CGH) assays, we investigated their intragenomic variations. The W chromosomes of both species did not accumulate higher amounts of both heterochromatin and repetitive sequences. However, while T. leucomelas showed a heterochromatin-poor W chromosome with a very complex evolutionary history, T. rufiventris showed a small and partially heterochromatic W chromosome that represents a differentiated version of its original autosomal complement (Z chromosome). The combined approach of CGH and sequential satDNA mapping suggest the occurrence of a former W-autosomal translocation event in T. leucomelas, which had an impact on the W chromosome in terms of sequence gains and losses. At the same time, an autosome, which is present in both males and females in a polymorphic state, lost sequences and integrated previously W-specific ones. This putative W-autosomal translocation, however, did not result in the emergence of a multiple-sex chromosome system. Instead, the generation of a neo-W chromosome suggests an unexpected evolutionary trajectory that deviates from the standard canonical model of sex chromosome evolution.

Chromosomal diversity in Crematogaster Lund, 1831 (Formicidae: Myrmicinae) from the Amazon rainforest.

Crematogaster Lund, 1831 is a speciose ant genus globally distributed and easily recognizable. Although biogeographical theories explain some variation among Neotropical Crematogaster, several taxonomical issues remain unresolved. While cytogenetic approaches can help to delimit species, cytogenetic data are only available for 18 taxa. In this study, classical and molecular cytogenetic analyses were performed on five Crematogaster species from the Brazilian Amazon to identify species-specific patterns. Two different cytotypes, both with 2n=22 chromosomes were observed in Crematogaster erecta Mayr, 1866, suggesting the presence of cryptic species, although with different karyotypic formulas. Crematogaster aff. erecta had 2n=28, while C. limata Smith, 1858, C. tenuicula Forel, 1904 and Crematogaster sp. had 2n = 38. The telomeric motif (TTAGG)n was found in all five species, and the (TCAGG)n motif was detected in the telomeres of C. limata. This peculiar motif was also detected in the centromeric regions of C. erecta cytotype I. The microsatellite (GA)n was dispersed in the chromosomes of all species studied which also had a single intrachromosomal rDNA site. The cytogenetic results revealed notable interspecific and intraspecific variation, which suggests different chromosomal rearrangements involved in the origin of these variations, also highlighting the taxonomic value of cytogenetic data on Crematogaster.

Satellite DNAs and the evolution of the multiple X1X2Y sex chromosomes in the wolf fish Hoplias malabaricus (Teleostei; Characiformes)

Multiple sex chromosomes usually arise from chromosomal rearrangements which involve ancestral sex chromosomes. There is a fundamental condition to be met for their long-term fixation: the meiosis must function, leading to the stability of the emerged system, mainly concerning the segregation of the sex multivalent. Here, we sought to analyze the degree of differentiation and meiotic pairing properties in the selected fish multiple sex chromosome system present in the wolf-fish Hoplias malabaricus (HMA). This species complex encompasses seven known karyotype forms (karyomorphs) where the karyomorph C (HMA-C) exhibits a nascent XY sex chromosomes from which the multiple X1X2Y system evolved in karyomorph HMA-D via a Y-autosome fusion. We combined genomic and cytogenetic approaches to analyze the satellite DNA (satDNA) content in the genome of HMA-D karyomorph and to investigate its potential contribution to X1X2Y sex chromosome differentiation. We revealed 56 satDNA monomers of which the majority was AT-rich and with repeat units longer than 100 bp. Seven out of 18 satDNA families chosen for chromosomal mapping by fluorescence in situ hybridization (FISH) formed detectable accumulation in at least one of the three sex chromosomes (X1, X2 and neo-Y). Nine satDNA monomers showed only two hybridization signals limited to HMA-D autosomes, and the two remaining ones provided no visible FISH signals. Out of seven satDNAs located on the HMA-D sex chromosomes, five mapped also to XY chromosomes of HMA-C. We showed that after the autosome-Y fusion event, the neo-Y chromosome has not substantially accumulated or eliminated satDNA sequences except for minor changes in the centromere-proximal region. Finally, based on the obtained FISHpatterns, we speculate on the possible contribution of satDNA to sex trivalent pairing and segregation.

Comparative molecular and conventional cytogenetic analyses of three species of Rhinella (Anura; Bufonidae).

The genus Rhinella corresponds to a group of anurans characterized by numerous taxonomic and systemic challenges, leading to their organization into species complexes. Cytogenetic data for this genus thus far are limited to the diploid number and chromosome morphology, which remain highly conserved among the species. In this study, we analyse the karyotypes of three species of the genus Rhinella (Rhinella granulosa, Rhinella margaritifera, and Rhinella marina) using both classical (conventional staining and C-banding) and molecular (FISH-fluorescence in situ hybridization with 18S rDNA, telomeric sequences, and microsatellite probes) cytogenetic approaches. The aim of this study is to provide data that can reveal variations in the distribution of repetitive sequences that can contribute to understanding karyotypic diversification in these species. The results revealed a conserved karyotype across the species, with 2n = 22 and FN = 44, with metacentric and submetacentric chromosomes. C-banding revealed heterochromatic blocks in the pericentromeric region for all species, with a proximal block on the long arms of pairs 3 and 6 in R. marina and on the short arms of pairs 4 and 6 in R. margaritifera. Additionally, 18S rDNA probes hybridized to pair 5 in R. granulosa, to pair 7 in R. marina, and to pair 10 in R. margaritifera. Telomeric sequence probes displayed signals exclusively in the distal region of the chromosomes, while microsatellite DNA probes showed species-specific patterns. These findings indicate that despite a conserved karyotypical macrostructure, chromosomal differences exist among the species due to the accumulation of repetitive sequences. This variation may be attributed to chromosome rearrangements or differential accumulation of these sequences, highlighting the dynamic role of repetitive sequences in the chromosomal evolution of Rhinella species. Ultimately, this study emphasizes the importance of the role of repetitive DNAs in chromosomal rearrangements to elucidate the evolutionary mechanisms leading to independent diversification in the distinct phylogenetic groups of Rhinella.

Long-Term, Non-Invasive Detection of Low-Dose Ionizing Radiation Exposure

Ionizing radiation induces complex changes in cells and tissues. The conventional approach to biological dosimetry has been to integrate physical and clinical measurements to optimize dose assessment. Molecular biodosimetry is an effective strategy to monitor radiation exposure and hematologic, cytogenetic, protein and transcript-based approaches have been developed to increase dose estimation accuracy. However, these approaches are invasive, time-consuming, have limited effectiveness over time, and importantly do not accurately inform on low dose radiation exposures. Therefore, novel, non-invasive, biomarkers are required that can overcome these limitations. We developed a pipeline that employs Fourier transform infrared (FTIR) spectroscopy in the mid-infrared spectrum to identify a signature of low dose ionizing radiation exposure in mouse ear pinnae over time. Two cohorts of C57BL/6J and one cohort of BALB/c mice were followed for ninety days after total body X-ray exposures of 10, 50, 100 or 200 cGy. The stratum corneum layer of the ear pinnae were repeatedly measured with attenuated total reflectance - focal plane array (ATR-FPA)-FTIR at 5, 14, 21, 49 and 90 days after radiation exposure. We found statistically significant discriminative power for all doses and all tested time-points out to 90 days after exposure. Classification accuracy was maximized when testing 14 days after exposure (specificity > 0.9 with a sensitivity threshold of 0.9) and dropped by roughly 30% sensitivity at 90 days. Since only hundreds of samples were required to learn highly discriminative signatures, developing human-relevant diagnostic capabilities is likely feasible and this non-invasive procedure points toward future non-invasive biodosimetry applications at population scales.

Transferring an Adult-Plant Stripe-Rust Resistance Gene Yr7VS from Chromosome 7V of Dasypyrum villosum (L.) to Bread Wheat.

Stripe rust (Puccinia striiformis West. f.sp. tritici, Pst) is a destructive disease that seriously threatens wheat production globally. Exploring novel resistance genes for use in wheat breeding is an urgent need, as continuous Pst evolution frequently leads to a breakdown of host resistance. Here, we identified a set of wheat-Dasypyrum villosum 01I139 (V#6) disomic introgression lines for the purpose of determining their responses to a mixture of Pst isolates CYR32, CYR33 and CYR34 at both seedling and adult-plant stages. The results showed that all introgression lines exhibited high susceptibility at the seedling stage, with infection-type (IT) scores in the range of 6-8, whereas, for chromosomes 5V#6 and 7V#6, disomic addition lines NAU5V#6-1 and NAU7V#6-1 displayed high resistance at the adult-plant stage, indicating that adult-plant resistance (APR) genes were located on them. Further, in order to transfer the stripe-rust resistance on chromosome 7V#6, four new wheat-D. villosum introgression lines were identified, by the use of molecular cytogenetic approaches, from the self-pollinated seeds of 7D and 7V#6, in double monosomic line NAU7V#6-2. Among them, NAU7V#6-3 and NAU7V#6-4 were t7V#6L and t7V#6S monosomic addition lines, and NAU7V#6-5 and NAU7V#6-6 were homozygous T7DS·7V#6L and T7DL·7V#6S whole-arm translocation lines. Stripe-rust tests and genetic analyses of chromosome 7V#6 introgression lines revealed a dominant APR gene designated as Yr7VS on the chromosome arm 7V#6S. Comparison with the homozygous T7DL·7V#6S translocation line and the recurrent parent NAU0686 showed no significant differences in yield-related traits. Thus, T7DL·7V#6S whole-arm translocation with the APR gene Yr7VS provided a valuable germplasm for breeding for resistance.

Evaluation of DNA damage through cytogenetic approach in smokers and vapers with and without nicotine compared with control group

The use of tobacco and electronic cigarettes is harmful to health and can cause genetic damage, increasing the risk of cancer and other serious diseases. Although e-cigarettes contain fewer toxic chemicals than conventional tobacco, recent studies have shown that the vapor caused by burning produced by e-cigarettes can also be toxic and carcinogenic. Various studies have found that those exposed to tobacco and vaping have significantly higher levels of damage to their DNA in different types of cells and tissues. Evidence has accumulated that e-cigarette vaping can alter cellular functions and DNA itself, increasing the risk of cancer and aging. The present work evaluatesthe cytogenetic damage in individuals exposed to conventional cigarette vapors by burning with nicotine and vapors without nicotine, compared with a non-smoking population. The study included participants with an average age of 30 years (+/-10), with a majority of men representing 70% of the sample. The Chi-square test found no significant statistical differences between the men and women exposed (p<0.05). The results of chromosomal fragility found in the four groups studied (control group, conventional smokers, vapers with nicotine and vapers without nicotine) showed breaks and gaps in one or both of the chromatids in all exposed individuals, with highly significant statistical differences (p<0.001) compared to the unexposed control group. No statistically significant differences were found between the group of conventional smokers and the vapers with and without nicotine, nor between the two types of vapers (p>0.05). In conclusion, cytogenetic evidence of DNA damage produced by vaping is shown in the same proportions as a normal cigarette. This will significantly impact public health, which must be considered in preventive actions. Keywords: cigarettes, vaping, chromosome breakage, DNA damage

Evaluation of DNA damage through cytogenetic approach in smokers and vapers with and without nicotine compared with control group

The use of tobacco and electronic cigarettes is harmful to health and can cause genetic damage, increasing the risk of cancer and other serious diseases. Although e-cigarettes contain fewer toxic chemicals than conventional tobacco, recent studies have shown that the vapor caused by burning produced by e-cigarettes can also be toxic and carcinogenic. Various studies have found that those exposed to tobacco and vaping have significantly higher levels of damage to their DNA in different types of cells and tissues. Evidence has accumulated that e-cigarette vaping can alter cellular functions and DNA itself, increasing the risk of cancer and aging. The present work evaluatesthe cytogenetic damage in individuals exposed to conventional cigarette vapors by burning with nicotine and vapors without nicotine, compared with a non-smoking population. The study included participants with an average age of 30 years (+/-10), with a majority of men representing 70% of the sample. The Chi-square test found no significant statistical differences between the men and women exposed (p<0.05). The results of chromosomal fragility found in the four groups studied (control group, conventional smokers, vapers with nicotine and vapers without nicotine) showed breaks and gaps in one or both of the chromatids in all exposed individuals, with highly significant statistical differences (p<0.001) compared to the unexposed control group. No statistically significant differences were found between the group of conventional smokers and the vapers with and without nicotine, nor between the two types of vapers (p>0.05). In conclusion, cytogenetic evidence of DNA damage produced by vaping is shown in the same proportions as a normal cigarette. This will significantly impact public health, which must be considered in preventive actions. Keywords: cigarettes, vaping, chromosome breakage, DNA damage

The First Identification of Homomorphic XY Sex Chromosomes by Integrating Cytogenetic and Transcriptomic Approaches in Plestiodon elegans (Scincidae)

The sex chromosomes of skinks are usually poorly differentiated and hardly distinguished by cytogenetic methods. Therefore, identifying sex chromosomes in species lacking easily recognizable heteromorphic sex chromosomes is necessary to fully understand sex chromosome diversity. In this paper, we employed cytogenetics, sex quantification of genes, and transcriptomic approaches to characterize the sex chromosomes in Plestiodon elegans. Cytogenetic examination of metaphases revealed a diploid number of 2n = 26, consisting of 12 macrochromosomes and 14 microchromosomes, with no significant heteromorphic chromosome pairs, speculating that the sex chromosomes may be homomorphic or poorly differentiated. The results of the sex quantification of genes showed that Calumenin (calu), COPI coat complex subunit γ 2 (copg2), and Smoothened (smo) were at half the dose in males as in females, suggesting that they are on the X chromosome. Transcriptomic data analysis from the gonads yielded the excess expression male-specific genes (n = 16), in which five PCR molecular markers were developed. Restricting the observed heterozygosity to males suggests the presence of homomorphic sex chromosomes in P. elegans, XX/XY. This is the first breakthrough in the study of the sex chromosomes of Plestiodon.

Genomic Characterization of Partial Tandem Duplication Involving the KMT2A Gene in Adult Acute Myeloid Leukemia.

Gene rearrangements affecting KMT2A are frequent in acute myeloid leukemia (AML) and are often associated with a poor prognosis. KMT2A gene fusions are often detected by chromosome banding analysis and confirmed by fluorescence in situ hybridization. However, small intragenic insertions, termed KMT2A partial tandem duplication (KMT2A-PTD), are particularly challenging to detect using standard molecular and cytogenetic approaches. We have validated the use of a custom hybrid-capture-based next-generation sequencing (NGS) panel for comprehensive profiling of AML patients seen at our institution. This NGS panel targets the entire consensus coding DNA sequence of KMT2A. To deduce the presence of a KMT2A-PTD, we used the relative ratio of KMT2A exons coverage. We sought to corroborate the KMT2A-PTD NGS results using (1) multiplex-ligation probe amplification (MLPA) and (2) optical genome mapping (OGM). We analyzed 932 AML cases and identified 41 individuals harboring a KMT2A-PTD. MLPA, NGS, and OGM confirmed the presence of a KMT2A-PTD in 22 of the cases analyzed where orthogonal testing was possible. The two false-positive KMT2A-PTD calls by NGS could be explained by the presence of cryptic structural variants impacting KMT2A and interfering with KMT2A-PTD analysis. OGM revealed the nature of these previously undetected gene rearrangements in KMT2A, while MLPA yielded inconclusive results. MLPA analysis for KMT2A-PTD is limited to exon 4, whereas NGS and OGM resolved KMT2A-PTD sizes and copy number levels. KMT2A-PTDs are complex gene rearrangements that cannot be fully ascertained using a single genomic platform. MLPA, NGS panels, and OGM are complementary technologies applied in standard-of-care testing for AML patients. MLPA and NGS panels are designed for targeted copy number analysis; however, our results showed that integration of concurrent genomic alterations is needed for accurate KMT2A-PTD identification. Unbalanced chromosomal rearrangements overlapping with KMT2A can interfere with the diagnostic sensitivity and specificity of copy-number-based KMT2A-PTD detection methodologies.

Evolution of bird sex chromosomes: a cytogenomic approach in Palaeognathae species

BackgroundDifferent patterns of sex chromosome differentiation are seen in Palaeognathae birds, a lineage that includes the ratites (Struthioniformes, Rheiformes, Apterygiformes, Casuariiformes, and the sister group Tinamiformes). While some Tinamiform species have well-differentiated W chromosomes, both Z and W of all the flightless ratites are still morphologically undifferentiated. Here, we conducted a comprehensive analysis of the ZW differentiation in birds using a combination of cytogenetic, genomic, and bioinformatic approaches. The whole set of satDNAs from the emu (Dromaius novaehollandiae) was described and characterized. Furthermore, we examined the in situ locations of these satDNAs alongside several microsatellite repeats and carried out Comparative Genomic Hybridizations in two related species: the greater rhea (Rhea americana) and the tataupa tinamou (Crypturellus tataupa).ResultsFrom the 24 satDNA families identified (which represent the greatest diversity of satDNAs ever uncovered in any bird species), only three of them were found to accumulate on the emu’s sex chromosomes, with no discernible accumulation observed on the W chromosome. The W chromosomes of both the greater rhea and the emu did not exhibit a significant buildup of either C-positive heterochromatin or repetitive DNAs, indicating their large undifferentiation both at morphological and molecular levels. In contrast, the tataupa tinamou has a highly differentiated W chromosome that accumulates several DNA repeats.ConclusionThe findings provide new information on the architecture of the avian genome and an inside look at the starting points of sex chromosome differentiation in birds.

A survey of chromosomal instability measures across mechanistic models

Chromosomal instability (CIN) is the persistent reshuffling of cancer karyotypes via chromosome mis-segregation during cell division. In cancer, CIN exists at varying levels that have differential effects on tumor progression. However, mis-segregation rates remain challenging to assess in human cancer despite an array of available measures. We evaluated measures of CIN by comparing quantitative methods using specific, inducible phenotypic CIN models of chromosome bridges, pseudobipolar spindles, multipolar spindles, and polar chromosomes. For each, we measured CIN fixed and timelapse fluorescence microscopy, chromosome spreads, six-centromere FISH, bulk transcriptomics, and single-cell DNA sequencing (scDNAseq). As expected, microscopy of tumor cells in live and fixed samples significantly correlated (R = 0.72; P < 0.001) and sensitively detect CIN. Cytogenetics approaches include chromosome spreads and 6-centromere FISH, which also significantly correlate (R = 0.76; P < 0.001) but had limited sensitivity for lower rates of CIN. Bulk genomic DNA signatures and bulk transcriptomic scores, CIN70 and HET70, did not detect CIN. By contrast, scDNAseq detects CIN with high sensitivity, and significantly correlates with imaging methods (R = 0.82; P < 0.001). In summary, single-cell methods such as imaging, cytogenetics, and scDNAseq can measure CIN, with the latter being the most comprehensive method accessible to clinical samples. To facilitate the comparison of CIN rates between phenotypes and methods, we propose a standardized unit of CIN: Mis-segregations per Diploid Division. This systematic analysis of common CIN measures highlights the superiority of single-cell methods and provides guidance for measuring CIN in the clinical setting.

Evaluation of DNA damage through cytogenetic approach in smokers and vapers with and without nicotine compared with control group

The use of tobacco and electronic cigarettes is harmful to health and can cause genetic damage, increasing the risk of cancer and other serious diseases. Although e-cigarettes contain fewer toxic chemicals than conventional tobacco, recent studies have shown that the vapor caused by burning produced by e-cigarettes can also be toxic and carcinogenic. Various studies have found that those exposed to tobacco and vaping have significantly higher levels of damage to their DNA in different types of cells and tissues. Evidence has accumulated that e-cigarette vaping can alter cellular functions and DNA itself, increasing the risk of cancer and aging. The present work evaluatesthe cytogenetic damage in individuals exposed to conventional cigarette vapors by burning with nicotine and vapors without nicotine, compared with a non-smoking population. The study included participants with an average age of 30 years (+/-10), with a majority of men representing 70% of the sample. The Chi-square test found no significant statistical differences between the men and women exposed (p&lt;0.05). The results of chromosomal fragility found in the four groups studied (control group, conventional smokers, vapers with nicotine and vapers without nicotine) showed breaks and gaps in one or both of the chromatids in all exposed individuals, with highly significant statistical differences (p&lt;0.001) compared to the unexposed control group. No statistically significant differences were found between the group of conventional smokers and the vapers with and without nicotine, nor between the two types of vapers (p&gt;0.05). In conclusion, cytogenetic evidence of DNA damage produced by vaping is shown in the same proportions as a normal cigarette. This will significantly impact public health, which must be considered in preventive actions. Keywords: cigarettes, vaping, chromosome breakage, DNA damage

Cytomolecular diversity among Vigna Savi (Leguminosae) subgenera.

The genus Vigna (Leguminosae) comprises about 150 species grouped into five subgenera. The present study aimed to improve the understanding of karyotype diversity and evolution in Vigna, using new and previously published data through different cytogenetic and DNA content approaches. In the Vigna subgenera, we observed a random distribution of rDNA patterns. The 35S rDNA varied in position, from terminal to proximal, and in number, ranging from one (V. aconitifolia, V. subg. Ceratotropis) to seven pairs (V. unguiculata subsp. unguiculata, V. subg. Vigna). On the other hand, the number of 5S rDNA was conserved (one or two pairs), except for V. radiata (V. subg. Ceratotropis), which had three pairs. Genome size was relatively conserved within the genus, ranging from 1C = 0.43 to 0.70pg in V. oblongifolia and V. unguiculata subsp. unguiculata, respectively, both belonging to V. subg. Vigna. However, we observed a positive correlation between DNA content and the number of 35S rDNA sites. In addition, data from chromosome-specific BAC-FISH suggest that the ancestral 35S rDNA locus is conserved on chromosome 6 within Vigna. Considering the rapid diversification in the number and position of rDNA sites, such conservation is surprising and suggests that additional sites may have spread out from this ancestral locus.

Satellitome Analysis in the Southern Lapwing (Vanellus chilensis) Genome: Implications for SatDNA Evolution in Charadriiform Birds.

Vanellus (Charadriidae; Charadriiformes) comprises around 20 species commonly referred to as lapwings. In this study, by integrating cytogenetic and genomic approaches, we assessed the satellite DNA (satDNA) composition of one typical species, Vanellus chilensis, with a highly conserved karyotype. We additionally underlined its role in the evolution, structure, and differentiation process of the present ZW sex chromosome system. Seven distinct satellite DNA families were identified within its genome, accumulating on the centromeres, microchromosomes, and the W chromosome. However, these identified satellite DNA families were not found in two other Charadriiformes members, namely Jacana jacana and Calidris canutus. The hybridization of microsatellite sequences revealed the presence of a few repetitive sequences in V. chilensis, with only two out of sixteen displaying positive hybridization signals. Overall, our results contribute to understanding the genomic organization and satDNA evolution in Charadriiform birds.

Comparative cytogenetics of the families Pteromalidae and Spalangiidae – a review

AbstractAlthough chromosomes of only 20 members of Pteromalidae sensu lato (s.l.), which belong to the families Pteromalidae sensu stricto (hence Pteromalidae) and Spalangiidae, are studied up to now, the accumulated cytogenetic information has important implications for taxonomic and evolutionary studies of these parasitoids. Within the former family, which includes the overwhelming majority of karyotypically studied species of Pteromalidae s.l., the known haploid chromosome number (n) can vary from 4 to 7 in Pachycrepoideus vindemmiae (Rondani) and Anisopteromalus calandrae (Howard), respectively, with a clear mode at n = 5. Among these parasitoids, Nasonia vitripennis (Walker) is the most thoroughly studied species in terms of modern cytogenetic techniques. On the contrary, chromosomes of only two members of Spalangiidae are known, both belonging to the Spalangia endius Walker complex, with n = 4 and 6. A few cosmopolitan cryptic species of the family Pteromalidae were studied using cytogenetic approaches. Specifically, Anisopteromalus quinarius Gokhman et Baur, a newly described parasitoid of stored‐product pests with n = 5, was initially separated from A. calandrae based on its different chromosome number. Another pteromalid with a similar biology, Lariophagus distinguendus (Förster), was also found to harbor two cryptic species with n = 5 and 6. Moreover, a morphometric study of these karyotypes suggested that the largest chromosome in the set with n = 5 has resulted from a fusion of two particular chromosomes in the karyotype with n = 6, and this hypothesis was confirmed using microdissection and whole‐chromosome painting. Recent cytogenetic and bioinformatic research also showed that N. vitripennis, Nasonia longicornis Darling, Nasonia giraulti Darling, Nasonia oneida Raychouhury et Desjardins, Muscidifurax uniraptor Kogan et Legner, and Trichomalopsis sarcophagae (Gahan) (all Pteromalidae) share the TTATTGGG telomeric motif with most other Chalcidoidea. Perspectives of the chromosome study of Pteromalidae s.l. are briefly discussed.

Monitoring Genomic Structural Rearrangements Resulting from Gene Editing.

The cytogenomics-based methodology of directional genomic hybridization (dGH) enables the detection and quantification of a more comprehensive spectrum of genomic structural variants than any other approach currently available, and importantly, does so on a single-cell basis. Thus, dGH is well-suited for testing and/or validating new advancements in CRISPR-Cas9 gene editing systems. In addition to aberrations detected by traditional cytogenetic approaches, the strand specificity of dGH facilitates detection of otherwise cryptic intra-chromosomal rearrangements, specifically small inversions. As such, dGH represents a powerful, high-resolution approach for the quantitative monitoring of potentially detrimental genomic structural rearrangements resulting from exposure to agents that induce DNA double-strand breaks (DSBs), including restriction endonucleases and ionizing radiations. For intentional genome editing strategies, it is critical that any undesired effects of DSBs induced either by the editing system itself or by mis-repair with other endogenous DSBs are recognized and minimized. In this paper, we discuss the application of dGH for assessing gene editing-associated structural variants and the potential heterogeneity of such rearrangements among cells within an edited population, highlighting its relevance to personalized medicine strategies.

A Next Generation Cytogenetic Approach to Elucidate the Complex Genetic Structure of Hematologic Neoplasms: Optical Genome Mapping

Hematolojik neoplazilerin tanısında ve tedavi yaklaşımlarının belirlenmesinde genetik anomalilerin rolü, konvansiyonel sitogenetik yöntemlerin gelişmesiyle birlikte paralel giden bir araştırma alanı olmuştur. Bu süreç moleküler sitogenetik ve moleküler genetik yöntemler ile farklı bir boyuta taşınmıştır. Bu teknikler çoğu zaman konvansiyonel sitogenetiğin eksik kalan yanlarını tamamlamış ancak yerini alamamıştır. Günümüze geldiğimizde ise Optik Genom Haritalama (OGH), konvansiyonel sitogenetiğin yerini alabilecek en muhtemel aday olarak görünmektedir. OGH, teorik olarak karyotip, FISH ve kromozomal mikroarray yöntemlerini aynı platformda buluşturan yeni nesil bir sitogenetik yaklaşım sergiler. Hücre kültürü gereksinimi ve amplifikasyon gerektirmemesi, hedefe spesifik bir yöntem olmaması en önemli artılarıdır. Klasik yöntemler ile saptanan tüm sayısal ve yapısal anomalileri saptayabilirken aynı zamanda kompleks karyotiplerin yapısını doğru bir şekilde aydınlatabilir. Ayrıca daha önce tanımlanmamış yeni anomaliler de tespit edebilir ki bu durum hematolojik neoplazilerde minimal rezidual hastalık takibinde yeni belirteçlerin bulunmasına olanak tanır. Tüm bunlar hematolojik neoplazili hastaların klinik yönetimine rehberlik edecek önemli unsurlardır. Ancak OGH, kırık noktaları tekrarlayan bölgeleri veya sentromer ve telomer bölgelerini ve psödootozomal bölgeleri içeren anomalileri tespit edemez. Bu yöntemin önemli bir dezavantajıdır. Yine de hematolojik neoplazilerin heterojen genetik yapısını tespit etmede genoma yeni bir sitogenetik yaklaşım sunar. Sonuç olarak sitogenetik açıdan baktığımızda hematolojik neoplazilerde tek başına yıllarca en önemli bilgi aracı olan kromozom analizi, gerekli teknik ve analitik iyileştirmelerin ardından belki de gelecekte yerini OGH’ye bırakacaktır.

Epigenetic histone H3 phosphorylation marks discriminate between univalent- and bivalent-forming chromosomes during canina asymmetrical meiosis.

Dogroses (Rosa sect. Caninae) are mostly pentaploid, bearing 2n = 5x = 35 chromosomes in somatic cells. They evolved a unique form of asymmetrical meiosis characterized by two types of chromosomes: (1) chromosomes forming bivalents and distributed in the normal sexual way; and (2) chromosomes occurring as univalents and transferred by a female gamete only. In the mature pollen of pentaploid species, seven bivalent-derived chromosomes are transmitted to offspring, and 21 unpaired univalent chromosomes are eliminated during microsporogenesis. To discriminate between bivalent- and univalent-forming chromosomes, we studied histone H3 phosphorylation patterns regulating meiotic chromosome condensation and segregation. We analysed histone modification patterns during male canina meiosis in two representative dogrose species, 5x Rosa canina and 5x Rosa rubiginosa, by immunohistochemical and molecular cytogenetics approaches. Immunostaining of meiotic cells included α-tubulin, histone H3 phosphorylation (H3S10p, H3S28p and H3T3p) and methylation (H3K4me3 and H3K27me3) marks. In addition, fluorescent in situ hybridization was carried out with an 18S rDNA probe. In the first meiotic division, univalent chromosomes underwent equational division into chromatids, while homologues in bivalents were segregated as regular dyads. In diakinesis, bivalent chromosomes displayed strong H3 phosphorylation signals in proximal regions, spreading to the rest of the chromosome. In contrast, in univalents, the H3 phosphorylation signals were weaker, occurring mostly outside proximal regions largely overlapping with the H3K4me3 signals. Reduced phosphorylation was associated with relative under-condensation of the univalent chromosomes, particularly at early diakinesis. We hypothesize that the absence of pairing and/or recombination in univalent chromosomes negatively affects the histone H3 phosphorylation of their chromatin and perhaps the loading of meiotic-specific cohesins. This apparently destabilizes cohesion of sister chromatids, leading to their premature split in the first meiotic division.