LD-CNV: rapid and simple discovery of chromosomal translocations using linkage disequilibrium between copy number variable loci

Large scale structural variations, such as chromosomal translocations, can have profound effects on fitness and phenotype, but are difficult to identify and characterize. Here, we describe a simple and effective method aimed at identifying translocations using only the dosage of sequence reads mapped on the reference genome. We binned reads on genomic segments sized according to sequencing coverage and identified instances when copy number segregated in populations. For each dosage-polymorphic 1Mb bin, we tested independence, effectively an apparent linkage disequilibrium (LD), with other variable bins. In nine potato (Solanum tuberosum) dihaploid families translocations affecting pericentromeric regions were common and in two cases were due to genomic misassembly. In two populations, we found evidence for translocation affecting euchromatic arms. In cv. PI 310467, a non-reciprocal translocation between chromosome 7 and 8 resulted in a 5-3 copy number change affecting several Mb at the respective chromosome tips. In cv. “Alca Tarma”, the terminal arm of chromosome 4 translocated to the tip of chromosome 1. Using oligonucleotide-based fluorescent in situ hybridization painting probes (oligo-FISH), we tested and confirmed the predicted arrangement in PI 310467. In 192 natural accessions of Arabidopsis thaliana, dosage haplotypes tended to vary continuously and resulted in higher noise, while apparent LD between pericentromeric regions suggested the effect of repeats. This method, LD-CNV, should be useful in species where translocations are suspected because it tests linkage without the need for genotyping.

Chromosomal Characterization of Tripidium arundinaceum Revealed by Oligo-FISH

Sugarcane is of important economic value for producing sugar and bioethanol. Tripidium arundinaceum (old name: Erianthus arundinaceum) is an intergeneric wild species of sugarcane that has desirable resistance traits for improving sugarcane varieties. However, the scarcity of chromosome markers has hindered the cytogenetic study of T. arundinaceum. Here we applied maize chromosome painting probes (MCPs) to identify chromosomes in sorghum and T. arundinaceum using a repeated fluorescence in situ hybridization (FISH) system. Sequential FISH revealed that these MCPs can be used as reliable chromosome markers for T. arundinaceum, even though T. arundinaceum has diverged from maize over 18 MYs (million years). Using these MCPs, we identified T. arundinaceum chromosomes based on their sequence similarity compared to sorghum and labeled them 1 through 10. Then, the karyotype of T. arundinaceum was established by multiple oligo-FISH. Furthermore, FISH results revealed that 5S rDNA and 35S rDNA are localized on chromosomes 5 and 6, respectively, in T. arundinaceum. Altogether, these results represent an essential step for further cytogenetic research of T. arundinaceum in sugarcane breeding.

New Data on Comparative Cytogenetics of the Mouse-Like Hamsters (Calomyscus Thomas, 1905) from Iran and Turkmenistan

The taxonomy of the genus Calomyscus remains controversial. According to the latest systematics the genus includes eight species with great karyotypic variation. Here, we studied karyotypes of 14 Calomyscus individuals from different regions of Iran and Turkmenistan using a new set of chromosome painting probes from a Calomyscus sp. male (2n = 46, XY; Shahr-e-Kord-Soreshjan-Cheshme Maiak Province). We showed the retention of large syntenic blocks in karyotypes of individuals with identical chromosome numbers. The only rearrangement (fusion 2/21) differentiated C. elburzensis, C. mystax mystax, and Calomyscus sp. from Isfahan Province with 2n = 44 from karyotypes of C. bailwardi, Calomyscus sp. from Shahr-e-Kord, Chahar Mahal and Bakhtiari-Aloni, and Khuzestan-Izeh Provinces with 2n = 46. The individuals from Shahdad tunnel, Kerman Province with 2n = 51–52 demonstrated non-centric fissions of chromosomes 4, 5, and 6 of the 46-chromosomal form with the formation of separate small acrocentrics. A heteromorphic pair of chromosomes in a specimen with 2n = 51 resulted from a fusion of two autosomes. C-banding and chromomycin A3-DAPI staining after G-banding showed extensive heterochromatin variation between individuals.

LD-CNV: rapid and simple discovery of chromosomal translocations using linkage disequilibrium between copy number variable loci

Large scale structural variations, such as chromosomal translocations, can have profound effects on fitness and phenotype, but are difficult to identify and characterize. Here, we describe a simple and effective method aimed at identifying translocations using only the dosage of sequence reads mapped on the reference genome. We binned reads on genomic segments sized according to sequencing coverage and identified instances when copy number segregated in populations. For each dosage-polymorphic 1Mb bin, we tested linkage disequilibrium with other variable bins. In nine potato (Solanum tuberosum) dihaploid families translocations affecting pericentromeric regions were common and in two cases were due to genomic misassembly. In two populations, we found evidence for translocation affecting euchromatic arms. In cv. PI 310467, a non-reciprocal translocation between chromosome 7 and 8 resulted in a 5-3 copy number change affecting several Mb at the respective chromosome tips. In cv. Alca Tarma the terminal arm of chromosome 4 translocated to the tip of chromosome 1. Using oligonucleotide-based fluorescent in situ hybridization painting probes (oligo-FISH), we tested and confirmed the predicted arrangement in PI 310467. In 192 natural accessions of Arabidopsis thaliana, dosage haplotypes tended to vary continuously and resulted in higher noise, but we identified pericentromeric LD suggesting the effect of repeats. This method should be useful in species where translocations are suspected because it tests linkage without the need for genotyping.

Identification of satellited markers by microdissection and fluorescence in situ hybridization: a clinical case of isodicentric chromosome 22

Background The presence of small supernumerary marker chromosomes (sSMCs) in a karyotype leads to diagnostic questions because the resulting extra material may cause abnormal development depending on the origin of the duplication/triplication. Because SMCs are so small, their origin cannot be determined by conventional cytogenetic techniques, and new molecular cytogenetic methods are necessary. Here, we applied a target approach to chromosome rearrangement analysis by isolating a chromosome of interest via microdissection and using it in fluorescence in situ hybridization (FISH) as a probe in combination with whole-chromosome painting probes. This approach allows to identify origins of both the euchromatin and repeat-rich regions of a marker. Case presentation We report a case of an adult male with congenital atresia of the rectum and anus, anotia, and atresia of the external auditory canal along with hearing loss. Karyotyping and FISH analysis with whole-chromosome painting probes of acrocentric chromosomes and the constructed microdissection library of the marker chromosome reliably identified an additional chromosome in some metaphases: mos 47,XY,+idic(22)(q11.2)[14]/46,XY [23]. Conclusion We propose to use whole-chromosome libraries and microdissected chromosomes in FISH to identify SMCs enriched with repeated sequences. We show that the methodology is successful in identifying the composition of a satellited marker chromosome.

Chromosome Abnormalities and Fertility in Domestic Bovids: A Review

After discovering the Robertsonian translocation rob(1;29) in Swedish red cattle and demonstrating its harmful effect on fertility, the cytogenetics applied to domestic animals have been widely expanded in many laboratories in order to find relationships between chromosome abnormalities and their phenotypic effects on animal production. Numerical abnormalities involving autosomes have been rarely reported, as they present abnormal animal phenotypes quickly eliminated by breeders. In contrast, numerical sex chromosome abnormalities and structural chromosome anomalies have been more frequently detected in domestic bovids because they are often not phenotypically visible to breeders. For this reason, these chromosome abnormalities, without a cytogenetic control, escape selection, with subsequent harmful effects on fertility, especially in female carriers. Chromosome abnormalities can also be easily spread through the offspring, especially when using artificial insemination. The advent of chromosome banding and FISH-mapping techniques with specific molecular markers (or chromosome-painting probes) has led to the development of powerful tools for cytogeneticists in their daily work. With these tools, they can identify the chromosomes involved in abnormalities, even when the banding pattern resolution is low (as has been the case in many published papers, especially in the past). Indeed, clinical cytogenetics remains an essential step in the genetic improvement of livestock.

Chromosome Painting in Cultivated Bananas and Their Wild Relatives (Musa spp.) Reveals Differences in Chromosome Structure

Edible banana cultivars are diploid, triploid, or tetraploid hybrids, which originated by natural cross hybridization between subspecies of diploid Musa acuminata, or between M. acuminata and diploid Musa balbisiana. The participation of two other wild diploid species Musa schizocarpa and Musa textilis was also indicated by molecular studies. The fusion of gametes with structurally different chromosome sets may give rise to progenies with structural chromosome heterozygosity and reduced fertility due to aberrant chromosome pairing and unbalanced chromosome segregation. Only a few translocations have been classified on the genomic level so far, and a comprehensive molecular cytogenetic characterization of cultivars and species of the family Musaceae is still lacking. Fluorescence in situ hybridization (FISH) with chromosome-arm-specific oligo painting probes was used for comparative karyotype analysis in a set of wild Musa species and edible banana clones. The results revealed large differences in chromosome structure, discriminating individual accessions. These results permitted the identification of putative progenitors of cultivated clones and clarified the genomic constitution and evolution of aneuploid banana clones, which seem to be common among the polyploid banana accessions. New insights into the chromosome organization and structural chromosome changes will be a valuable asset in breeding programs, particularly in the selection of appropriate parents for cross hybridization.

Cytokinesis-Block Micronucleus Cytome Assay Evolution into a More Comprehensive Method to Measure Chromosomal Instability

This review describes the cytokinesis-block micronucleus (CBMN) cytome assay and its evolution into a molecular cytogenetic method of chromosomal instability (CIN). Micronuclei (MNi) originate from whole chromosomes or chromosome fragments that fail to segregate to the poles of the cell during mitosis. These lagging chromosomes are excluded from the daughter nuclei and are enveloped in their own membrane to form MNi. The CBMN assay was developed to allow MNi to be scored exclusively in once-divided binucleated cells, which enables accurate measurement of chromosome breakage or loss without confounding by non-dividing cells that cannot express MNi. The CBMN assay can be applied to cell lines in vitro and cells such as lymphocytes that can be stimulated to divide ex vivo. In the CBMN assay, other CIN biomarkers such as nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) are also measured. Use of centromere, telomere, and chromosome painting probes provides further insights into the mechanisms through which MNi, NPBs and NBUDs originate. Measurement of MNi is also important because entrapment within a micronucleus may cause chromosomes to shatter and, after nuclear reintegration, become rearranged. Additionally, leakage of DNA from MNi can stimulate inflammation via the cyclic GMP-AMP Synthase—Stimulator of Interferon Genes (cGAS-STING) DNA sensing mechanism of the innate immune system.

Chromosome painting in cultivated banana and their wild relatives (Musa spp.) reveals differences in chromosome structure

Edible banana cultivars are diploid, triploid or tetraploid hybrids which originated by natural cross hybridization between subspecies of diploid Musa acuminata, or between M. acuminata and diploid M. balbisiana. Participation of two other wild diploid species M. schizocarpa and M. textilis was also indicated by molecular studies. Fusion of gametes with structurally different chromosome sets may give rise to progenies with structural chromosome heterozygosity and reduced fertility due to aberrant chromosome pairing and unbalanced chromosome segregation. Only a few translocations have been classified on the genomic level so far and a comprehensive molecular cytogenetic characterization of cultivars and species of the family Musaceae is still lacking. FISH with chromosome-arm specific oligo painting probes was used for comparative karyotype analysis in a set of wild Musa species and edible banana clones. The results revealed large differences in chromosome structure discriminating individual accessions. These results permitted identification of putative progenitors of cultivated clones and clarified genomic constitution and evolution of aneuploid banana clones, which seem to be common among the polyploid banana accessions. New insights into the chromosome organization and structural chromosome changes will be a valuable asset in breeding programs, particularly in selection of appropriate parents for cross hybridization.HighlightOligo painting FISH revealed chromosomal translocations in subspecies of Musa acuminata (A genome), their intra-specific hybrids as well as in M. balbisiana (B genome) and in interspecific hybrid clones originating from cross hybridization between M. acuminata and M. balbisiana