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For peanut, the lack of stable cytological markers is a barrier to tracking specific chromosomes, elucidating the genetic relationships between genomes and identifying chromosomal variations. Chromosome mapping using single-copy oligonucleotide (oligo) probe libraries has unique advantages for identifying homologous chromosomes and chromosomal rearrangements. In this study, we developed two whole-chromosome single-copy oligo probe libraries, LS-7A and LS-8A, based on the reference genome sequences of chromosomes 7A and 8A of Arachis duranensis . Fluorescence in situ hybridization (FISH) analysis confirmed that the libraries could specifically paint chromosomes 7 and 8. In addition, sequential FISH and electronic localization of LS-7A and LS-8A in A. duranensis ( AA) and A. ipaensis (BB) showed that chromosomes 7A and 8A contained translocations and inversions relative to chromosomes 7B and 8B. Analysis of the chromosomes of wild Arachis species using LS-8A confirmed that this library could accurately and effectively identify A genome species. Finally, LS-7A and LS-8A were used to paint the chromosomes of interspecific hybrids and their progenies, which verified the authenticity of the interspecific hybrids and identified a disomic addition line. This study provides a model for developing specific oligo probes to identify the structural variations of other chromosomes in Arachis and demonstrates the practical utility of LS-7A and LS-8A., Competing Interests: The authors declare that they have no competing interests.

Somatic Y chromosome loss in hematopoietic cells is associated with higher mortality in men. However, the status of the Y chromosome in cancer tissue is not fully known due to technical limitations, such as difficulties in labelling and sequencing DNA from the Y chromosome. We have developed a system to quantify Y chromosome gain or loss in patient-derived prostate cancer organoids. Using our system, we observed Y chromosome loss in 4 of the 13 (31%) patient-derived metastatic castration-resistant prostate cancer (mCRPC) organoids; interestingly, loss of Yq (long arm of the Y chromosome) was seen in 38% of patient-derived organoids. Additionally, potential associations were observed between mCRPC and Y chromosome nullisomy. The prevalence of Y chromosome loss was similar in primary and metastatic tissue, suggesting that Y chromosome loss is an early event in prostate cancer evolution and may not a result of drug resistance or organoid derivation. This study reports quantification of Y chromosome loss and gain in primary and metastatic prostate cancer tissue and lays the groundwork for further studies investigating the clinical relevance of Y chromosome loss or gain in mCRPC., Competing Interests: Sai Harisha Rajanala: No competing interests to declare. Romina Ghale: No competing interests to declare. Subhiksha Nandakumar: No competing interests to declare. Kalyani Chadalavada: No competing interests to declare. Gwo-Shu Mary Lee: No competing interests to declare. Konrad H. Stopsack: No competing interests to declare. Yu Chen: No competing interests to declare. Gouri J. Nanjangud: No competing interests to declare. Goutam Chakraborty: Has served as a scientific consultant for GuidePoint and received consultation fees. Philip W. Kantoff: The author has financial interest in Convergent Therapeutics, Inc., Context Therapeutics Inc., and ESSA Pharma Inc. He is a member of the Board of Directors for Convergent Therapeutics, Inc., Context Therapeutics Inc., and ESSA Pharma Inc. He is a consultant/member of the Scientific Advisory Board for Immunis.AI and PrognomIQ. The author has no other competing interests to declare. This does not alter our adherence to PLOS ONE policies on sharing data and materials., (Copyright: © 2024 Rajanala et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Introduction: Rhipidomys is the second most specious and the most widespread genus of the tribe Thomasomyini. Chromosomal data have been an important tool in the taxonomy of the group that presents low variability of diploid number (2n) and highly variable fundamental numbers (FNs). Despite such diversity, the genus has been studied mainly by classical and banding cytogenetic techniques., Methods: This study performed a comparative study between R. emiliae (2n = 44, FN = 52), R. macrurus (2n = 44, FN = 49), R. nitela (2n = 50, FN = 71), and R. mastacalis (2n = 44, FN = 72) using chromosome painting probes of two Oryzomyini species., Results: Our analysis revealed pericentric inversion as the main rearrangement involved in the karyotype evolution of the group, although tandem fusions/fissions were also detected. In addition, we detected eight syntenic associations exclusive of the genus Rhipidomys, and three syntenic associations shared between species of the tribe Thomasomyini and Oryzomyini., Conclusion: Comparative cytogenetic analysis by ZOO-FISH on genus Rhipidomys supports a pattern of chromosomal rearrangement already suggested by comparative G-banding. However, the results suggest that karyotype variability in the genus could also involve the occurrence of an evolutionary new centromere., (© 2024 S. Karger AG, Basel.)

Pelecaniformes is an order of waterbirds that exhibit diverse and distinct morphologies. Ibis, heron, pelican, hammerkop, and shoebill are included within the order. Despite their fascinating features, the phylogenetic relationships among the families within Pelecaniformes remain uncertain and pose challenges due to their complex evolutionary history. Their karyotypic evolution is another little-known aspect. Therefore, to shed light on the chromosomal rearrangements that have occurred during the evolution of Pelecaniformes, we have used whole macrochromosome probes from Gallus gallus (GGA) to show homologies on three species with different diploid numbers, namely Cochlearius cochlearius (2n = 74), Eudocimus ruber (2n = 66), and Syrigma sibilatrix (2n = 62). A fusion between GGA6 and GGA7 was found in C. cochlearius and S. sibilatrix. In S. sibilatrix the GGA8, GGA9 and GGA10 hybridized to the long arms of biarmed macrochromosomes, indicating fusions with microchromosomes. In E. ruber the GGA7 and GGA8 hybridized to the same chromosome pair. After comparing our painting results with previously published data, we show that distinct chromosomal rearrangements have occurred in different Pelecaniformes lineages. Our study provides new insight into the evolutionary history of Pelecaniformes and the chromosomal changes involving their macrochromosomes and microchromosomes that have taken place in different species within this order., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Seligmann et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Key Message: Chromosome-specific painting probes were developed to identify the individual chromosomes from 1 to 7E in Thinopyrum species and detect alien genetic material of the E genome in a wheat background. The E genome of Thinopyrum is closely related to the ABD genome of wheat (Triticum aestivum L.) and harbors genes conferring beneficial traits to wheat, including high yield, disease resistance, and unique end-use quality. Species of Thinopyrum vary from diploid (2n = 2x = 14) to decaploid (2n = 10x = 70), and chromosome structural variation and differentiation have arisen during polyploidization. To investigate the variation and evolution of the E genome, we developed a complete set of E genome-specific painting probes for identification of the individual chromosomes 1E to 7E based on the genome sequences of Th. elongatum (Host) D. R. Dewey and wheat. By using these new probes in oligonucleotide-based chromosome painting, we showed that Th. bessarabicum (PI 531711, E b E b ) has a close genetic relationship with diploid Th. elongatum (E e E e ), with five chromosomes (1E, 2E, 3E, 6E, and 7E) maintaining complete synteny in the two species except for a reciprocal translocation between 4 and 5E b . All 14 pairs of chromosomes of tetraploid Th. elongatum have maintained complete synteny with those of diploid Th. elongatum (Thy14), but the two sets of E genomes have diverged. This study also demonstrated that the E genome-specific painting probes are useful for rapid and effective detection of the alien genetic material of E genome in wheat-Thinopyrum derived lines., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Some types of chromosome aberrations are not easily identified by the traditional Giemsa staining. It usually needs some banding technique and skilled person's eye. Whole chromosome painting FISH probe can stain designated entire chromosomes or domains in metaphase chromosomes or interphase nuclei, respectively. It allows to visually identify translocations, deletions, or amplifications of specific chromosomes. Once chromosomes are stained, even non-skilled researchers can easily identify those chromosome aberrations. Whole chromosome painting FISH has higher sensitivity to detect chromosome aberrations. This chapter introduces the method for whole chromosome painting FISH staining., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

X-autosome translocation (XY 1 Y 2 ) has been reported in distinct groups of vertebrates suggesting that the rise of a multiple sex system within a species may act as a reproductive barrier and lead to speciation. The viability of this system has been linked with repetitive sequences located between sex and autosomal portions of the translocation. Herein, we investigate Oecomys auyantepui, using chromosome banding and Fluorescence In Situ Hybridization with telomeric and Hylaeamys megacephalus whole-chromosome probes, and phylogenetic reconstruction using mtDNA and nuDNA sequences. We describe an amended karyotype for O. auyantepui (2n = 64♀65♂/FNa = 84) and report for the first time a multiple sex system (XX/XY 1 Y 2 ) in Oryzomyini rodents. Molecular data recovered O. auyantepui as a monophyletic taxon with high support and cytogenetic data indicate that O. auyantepui may exist in two lineages recognized by distinct sex systems. The Neo-X exhibits repetitive sequences located between sex and autosomal portions, which would act as a boundary between these two segments. The G-banding comparisons of the Neo-X chromosomes of other Sigmodontinae taxa revealed a similar banding pattern, suggesting that the autosomal segment in the Neo-X can be shared among the Sigmodontinae lineages with a XY 1 Y 2 sex system., (© 2022. The Author(s).)

Karyotypes provide key cytogenetic information on the phylogenetic relationships and evolutionary origins in related eukaryotic species. Despite our knowledge of the chromosome numbers of sugarcane and its wild relatives, the chromosome composition and evolution among the species in the Saccharum complex have been elusive owing to the complex polyploidy and the large numbers of chromosomes of these species. Oligonucleotide-based chromosome painting has become a powerful tool of cytogenetic studies especially for plant species with large numbers of chromosomes. We developed oligo-based chromosome painting probes for all 10 chromosomes in Saccharum officinarum (2n = 8x = 80). The 10 painting probes generated robust fluorescence in situ hybridization signals in all plant species within the Saccharum complex, including species in the genera Saccharum, Miscanthus, Narenga and Erianthus. We conducted comparative chromosome analysis using the same set of probes among species from four different genera within the Saccharum complex. Excitingly, we discovered several novel cytotypes and chromosome rearrangements in these species. We discovered that fusion from two different chromosomes is a common type of chromosome rearrangement associated with the species in the Saccharum complex. Such fusion events changed the basic chromosome number and resulted in distinct allopolyploids in the Saccharum complex., (© 2021 The Authors. New Phytologist © 2021 New Phytologist Foundation.)

Chromosome karyotyping analysis is particularly useful in determining species relationships and the origin of polyploid species. Identification of individual chromosomes is the foundation for karyotype development. For Fragaria (strawberry) species, definitive identification of the individual chromosomes is extremely difficult because of their small size and similar shape. Here, we identified all chromosomes for 11 representative Fragaria species with different ploidy using a set of oligonucleotide-based probes developed in Fragaria vesca. Comprehensive molecular cytogenetic karyotypes were established based on the individually identified chromosomes. In addition, we used oligo probes to assign the 5S and 45S rDNA loci to specific chromosomes in 16 Fragaria species. We found that these Fragaria species maintained a remarkably conserved karyotype. No inter-chromosomal structural rearrangements at the cytological level were observed in any of the chromosomes among these species. Despite karyotypic stability and similarity, variations in the signal intensity of oligo probes were observed among the homologous chromosomes in several polyploid species. Moreover, most Fragaria species also showed differences in the distribution patterns of 45S and 5S rDNA. These data provide new insights into the origins of several polyploid Fragaria species., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Rhipidomys (Sigmodontinae, Thomasomyini) has 25 recognized species, with a wide distribution ranging from eastern Panama to northern Argentina. Cytogenetic data has been described for 13 species with 12 of them having 2n = 44 with a high level of autosomal fundamental number (FN) variation, ranging from 46 to 80, assigned to pericentric inversions. The species are grouped in groups with low FN (46-52) and high FN (72-80). In this work the karyotypes of Rhipidomys emiliae (2n = 44, FN = 50) and Rhipidomys mastacalis (2n = 44, FN = 74), were studied by classical cytogenetics and by fluorescence in situ hybridization using telomeric and whole chromosome probes (chromosome painting) of Hylaeamys megacephalus (HME). Chromosome painting revealed homology between 36 segments of REM and 37 of RMA. We tested the hypothesis that pericentric inversions are the predominant chromosomal rearrangements responsible for karyotypic divergence between these species, as proposed in literature. Our results show that the genomic diversification between the karyotypes of the two species resulted from translocations, centromeric repositioning and pericentric inversions. The chromosomal evolution in Rhipidomys was associated with karyotypical orthoselection. The HME probes revealed that seven syntenic probably ancestral blocks for Sigmodontinae are present in Rhipidomys. An additional syntenic block described here is suggested as part of the subfamily ancestral karyotype. We also define five synapomorphies that can be used as chromosomal signatures for Rhipidomys., Competing Interests: The authors have declared that no competing interests exist.

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.

Chacoan peccary (Catagonus wagneri, 2n=20) is the most endangered of three extant species of Tayassuidae. Its karyotype has been studied only by differential chromosome staining methods so far. To establish a comparative cytogenetic map of the peccary, we used cross-species hybridization with porcine (Sus scrofa, 2n=38) painting probes. Painting revealed 30 evolutionary conserved autosomal segments between pig and peccary. The q-arm of the submetacentric chromosome X is homologous to the porcine X chromosome, while the p-arm is composed of heterochromatin. Nucleolar organizer regions were detected on chromosomes 8 and 9 which are homologous to pig chromosomes 8 and 4/18, respectively. Fusions of chromosomes homologous to pig chromosomes 4/7 and 4/18 and fission of chromosome 7 are synapomorphic characters shared by Catagonus wagneri and Tayassu pecari but not by Pecari tajacu. Our results confirmed a high rate of karyotype evolution in Tayassuidae and a closer relationship of Catagonus wagneri with Tayassu pecari than with Pecari tajacu.

Background: Thamnophilidae birds are the result of a monophyletic radiation of insectivorous Passeriformes. They are a diverse group of 225 species and 45 genera and occur in lowlands and lower montane forests of Neotropics. Despite the large degree of diversity seen in this family, just four species of Thamnophilidae have been karyotyped with a diploid number ranging from 76 to 82 chromosomes. The karyotypic relationships within and between Thamnophilidae and another Passeriformes therefore remain poorly understood. Recent studies have identified the occurrence of intrachromosomal rearrangements in Passeriformes using in silico data and molecular cytogenetic tools. These results demonstrate that intrachromosomal rearrangements are more common in birds than previously thought and are likely to contribute to speciation events. With this in mind, we investigate the apparently conserved karyotype of Willisornis vidua, the Xingu Scale-backed Antbird, using a combination of molecular cytogenetic techniques including chromosome painting with probes derived from Gallus gallus (chicken) and Burhinus oedicnemus (stone curlew), combined with Bacterial Artificial Chromosome (BAC) probes derived from the same species. The goal was to investigate the occurrence of rearrangements in an apparently conserved karyotype in order to understand the evolutionary history and taxonomy of this species. In total, 78 BAC probes from the Gallus gallus and Taeniopygia guttata (the Zebra Finch) BAC libraries were tested, of which 40 were derived from Gallus gallus macrochromosomes 1-8, and 38 from microchromosomes 9-28., Results: The karyotype is similar to typical Passeriformes karyotypes, with a diploid number of 2n = 80. Our chromosome painting results show that most of the Gallus gallus chromosomes are conserved, except GGA-1, 2 and 4, with some rearrangements identified among macro- and microchromosomes. BAC mapping revealed many intrachromosomal rearrangements, mainly inversions, when comparing Willisornis vidua karyotype with Gallus gallus, and corroborates the fissions revealed by chromosome painting., Conclusions: Willisornis vidua presents multiple chromosomal rearrangements despite having a supposed conservative karyotype, demonstrating that our approach using a combination of FISH tools provides a higher resolution than previously obtained by chromosome painting alone. We also show that populations of Willisornis vidua appear conserved from a cytogenetic perspective, despite significant phylogeographic structure.

The differentiation of sex chromosomes is thought to be interrupted by relatively frequent sex chromosome turnover and/or occasional recombination between sex chromosomes (fountain-of-youth model) in some vertebrate groups as fishes, amphibians, and lizards. As a result, we observe the prevalence of homomorphic sex chromosomes in these groups. Here, we provide evidence for the loss of sex chromosome heteromorphism in the Amazonian frogs of the genus Engystomops, which harbors an intriguing history of sex chromosome evolution. In this species complex composed of two named species, two confirmed unnamed species, and up to three unconfirmed species, highly divergent karyotypes are present, and heteromorphic X and Y chromosomes were previously found in two species. We describe the karyotype of a lineage estimated to be the sister of all remaining Amazonian Engystomops (named Engystomops sp.) and perform chromosome painting techniques using one probe for the Y chromosome and one probe for the non-centromeric heterochromatic bands of the X chromosome of E. freibergi to compare three Engystomops karyotypes. The Y probe detected the Y chromosomes of E. freibergi and E. petersi and one homolog of chromosome pair 11 of Engystomops sp., suggesting their common evolutionary origin. The X probe showed no interspecific hybridization, revealing that X chromosome heterochromatin is strongly divergent among the studied species. In the light of the phylogenetic relationships, our data suggest that sex chromosome heteromorphism may have occurred early in the evolution of the Amazonian Engystomops and have been lost in two unnamed but confirmed candidate species.

Background: The Scolopacidae family (Suborder Scolopaci, Charadriiformes) is composed of sandpipers and snipes; these birds are long-distance migrants that show great diversity in their behavior and habitat use. Cytogenetic studies in the Scolopacidae family show the highest diploid numbers for order Charadriiformes. This work analyzes for the first time the karyotype of Actitis macularius by classic cytogenetics and chromosome painting., Results: The species has a diploid number of 92, composed mostly of telocentric pairs. This high 2n is greater than the proposed 80 for the avian ancestral putative karyotype (a common feature among Scolopaci), suggesting that fission rearrangements have formed smaller macrochromosomes and microchromosomes. Fluorescence in situ hybridization using Burhinus oedicnemus whole chromosome probes confirmed the fissions in pairs 1, 2, 3, 4 and 6 of macrochromosomes., Conclusion: Comparative analysis with other species of Charadriiformes studied by chromosome painting together with the molecular phylogenies for the order allowed us to raise hypotheses about the chromosomal evolution in suborder Scolopaci. From this, we can establish a clear idea of how chromosomal evolution occurred in this suborder.

The subfamily Lonchorhininae encompasses 6 species of sword-nosed bats (Lonchorhina) and is one of the most problematic lineages in the Neotropical leaf-nosed bats (Phyllostomidae) phylogeny. There are at least 5 different hypotheses to explain when the subfamily diverged from the remaining phyllostomids, but none with robust statistical support. Here, we generated a chromosome painting homology map of Lonchorhina aurita karyotype (2n = 32 and FN = 60) using whole-chromosome probes of Macrotus californicus (MCA; 2n = 40 and FN = 60). We placed the karyotype changes of L. aurita in a phylogenetic context to discuss the most likely branching position of Lonchorhininae based on karyotypic evolution. We show that L. aurita has a derived karyotype with 24 segments homologous to the 20 MCA chromosomes used as probes. Comparative analyses between 7 published painted bats species across 4 phyllostomid subfamilies (Macrotinae, Phyllostominae, Glossophaginae, and Lonchophyllinae) revealed that one inversion (MCA 4inv) and one fusion (MCA 17 + 18) are shared derived features between the karyotypes of L. aurita and species of Phyllostominae not yet observed in other bats. Our data show that chromosomal homology maps may contribute with new insights into a long-standing phylogenetic debate that has endured for decades., (© 2022 S. Karger AG, Basel.)

Chromosome painting is a useful technique for distinguishing specific chromosomes (fragments), elucidating the genetic relationships of different genomes or chromosomes, and identifying chromosomal rearrangements. The development of chromosome- or genome-specific probes is fundamental for chromosome painting. The possibility for developing such probes specifically painting homoeologous chromosomes in allopolyploid species has been questioned since that chromosomes belonging to the same homoeologous group share highly conserved sequences. In the present study, we attempted to construct a wheat chromosome 4D-specific oligo probe library by selecting 4D-specific sequences in reference genome of common wheat cv. Chinese Spring (CS, 2n = 6x = 42, AABBDD). The synthesized library contains 27,392 oligos. Oligo painting using the probe library confirmed its specificity, shown by that only chromosome 4D could be painted in three wheat genotypes and CS nulli-tetrasomic line N4AT4D. Oligo painting was successfully used to define the 4D breakpoints in CS deletion lines involving 4D and two wheat-Haynaldia villosa 4D-4V translocation lines. Thirteen wheat relatives and a Triticum durum-H. villosa amphiploid were used for oligo painting. Except the 4D in two Aegilops tauschii accessions, the 4M in Ae. comosa and 4U in Ae. umbellulata could be painted. In tetraploid Ae. ventricosa, both 4D and 4M could be painted; however, the signal intensity of 4M was less compared with 4D. No painted chromosome was observed for the other alien species. This indicated that the relationship among D/M/U was closer than that among D/A/B as well as D with genomes H/R/S s /S c /Y/P/N/J. Our successful development of 4D-specific oligo probe library may serve as a model for developing oligo probes specific for other homoeologous chromosomes.

Chromosome painting is a powerful technique for chromosome and genome studies. We developed a flexible chromosome painting technique based on multiplex PCR of a synthetic oligonucleotide (oligo) library in cucumber (Cucumis sativus L., 2n = 14). Each oligo in the library was associated with a universal as well as nested specific primers for amplification, which allow the generation of different probes from the same oligo library. We were also able to generate double-stranded labelled oligos, which produced much stronger signals than single-stranded labelled oligos, by amplification using fluorophore-conjugated primer pairs. Oligos covering cucumber chromosome 1 (Chr1) and chromosome 4 (Chr4) consisting of eight segments were synthesized in one library. Different oligo probes generated from the library painted the corresponding chromosomes/segments unambiguously, especially on pachytene chromosomes. This technique was then applied to study the homoeologous relationships among cucumber, C. hystrix and C. melo chromosomes based on cross-species chromosome painting using Chr4 probes. We demonstrated that the probe was feasible to detect interspecies chromosome homoeologous relationships and chromosomal rearrangement events. Based on its advantages and great convenience, we anticipate that this flexible oligo-painting technique has great potential for the studies of the structure, organization, and evolution of chromosomes in any species with a sequenced genome., (© 2019 The Authors The Plant Journal © 2019 John Wiley & Sons Ltd.)

Efficient and consistent chromosome identification is the foundation for successful cytogenetic studies. Fluorescent in situ hybridization (FISH) has been the most popular technique for chromosome identification in plants. Large insert genomic DNA clones, such as bacterial artificial chromosome (BAC) clones, and repetitive DNA sequences have been the most commonly used DNA probes for FISH. However, most of such traditional probes can only be used to identify a single chromosome or are too polymorphic to consistently identify the same chromosome in the target species. In contrast, FISH using oligonucleotide (oligo)-based probes is highly versatile. In this procedure, a large number of oligos specific to a chromosomal region, to an entire chromosome, or to multiple chromosomes are computationally identified, synthesized in parallel, and labeled as probes. In addition, each oligo probe can be used for thousands of FISH experiments and represents an infinite resource. In this chapter we describe a detailed protocol for amplification and labeling of oligo-based probes, relevant chromosome preparation, and FISH procedures.

The karyotype represents the basic genetic make-up of a eukaryotic species. Comparative cytogenetic analysis of related species based on individually identified chromosomes has been conducted in only a few plant groups and not yet in woody plants. We have developed a complete set of 19 chromosome painting probes based on the reference genome of the model woody plant Populus trichocarpa. Using sequential fluorescence in situ hybridization we were able to identify all poplar chromosomes in the same metaphase cells, which led to the development of poplar karyotypes based on individually identified chromosomes. We demonstrate that five Populus species, belonging to five different sections within Populus, have maintained a remarkably conserved karyotype. No inter-chromosomal structural rearrangements were observed on any of the 19 chromosomes among the five species. Thus, the chromosomal synteny in Populus has been remarkably maintained after nearly 14 million years of divergence. We propose that the karyotypes of woody species are more stable than those of herbaceous plants since it may take a longer period of time for woody plants to fix chromosome number or structural variants in natural populations., (© 2019 The Authors The Plant Journal © 2019 John Wiley & Sons Ltd.)

Visualization of meiotic chromatin from pollen mother cells has become an essential technique to study meiosis in the model plant Arabidopsis thaliana. Here we present an advanced cytogenetic method that combines improved immunocytology with chromosome painting, thereby generating a tool to quantitatively analyze localization of proteins to any given genomic region. Proteins involved in different processes such as DNA double-strand break formation and recombinational repair can be visualized on meiotic chromatin with the additional feature of assessing their abundance at specific chromosomal locations.

Karyotypes of two cryptic species of parasitoid Hymenoptera with n = 5 and 6 belonging to the Lariophagus distinguendus (Förster, 1841) complex, which includes cosmopolitan parasitoids of coleopteran stored-product pests, were studied using glass-needle based microdissection, reverse and cross-species fluorescence in situ hybridisation (FISH). This experiment strongly indicates that the largest metacentric chromosome in the karyotype with n = 5 originated from a particular fusion between the only acrocentric and a smaller metacentric chromosome of the set with n = 6, therefore confirming our previous hypothesis based on the karyotypic analysis using chromosome morphometrics. This study represents the first successful application of both microdissection and whole chromosome painting for the reconstruction of karyotypic rearrangements in closely related species of parasitoids, as well as in the order Hymenoptera in general., Competing Interests: The authors have declared that no competing interests exist.

Background: The Neacomys genus is predominantly found in the Amazon region, and belongs to the most diverse tribe of the Sigmodontinae subfamily (Rodentia, Cricetidae, Oryzomyini). The systematics of this genus and questions about its diversity and range have been investigated by morphological, molecular (Cytb and COI sequences) and karyotype analysis (classic cytogenetics and chromosome painting), which have revealed candidate species and new distribution areas. Here we analyzed four species of Neacomys by chromosome painting with Hylaeamys megacephalus (HME) whole-chromosome probes, and compared the results with two previously studied Neacomys species and with other taxa from Oryzomyini and Akodontini tribes that have been hybridized with HME probes. Maximum Parsimony (MP) analyses were performed with the PAUP and T.N.T. software packages, using a non-additive (unordered) multi-state character matrix, based on chromosomal morphology, number and syntenic blocks. We also compared the chromosomal phylogeny obtained in this study with molecular topologies (Cytb and COI) that included eastern Amazonian species of Neacomys, to define the phylogenetic relationships of these taxa., Results: The comparative chromosome painting analysis of the seven karyotypes of the six species of Neacomys shows that their diversity is due to 17 fusion/fission events and one translocation, pericentric inversions in four syntenic blocks, and constitutive heterochromatin (CH) amplification/deletion of six syntenic autosomal blocks plus the X chromosome. The chromosomal phylogeny is consistent with the molecular relationships of species of Neacomys. We describe new karyotypes and expand the distribution area for species from eastern Amazonia and detect complex rearrangements by chromosome painting among the karyotypes., Conclusions: Our phylogeny reflects the molecular relationships of the Akodontini and Oryzomyini taxa and supports the monophyly of Neacomys. This work presents new insights about the chromosomal evolution of this group, and we conclude that the karyotypic divergence is in accord with phylogenetic relationships.

Measurements of nuclear organization in asymmetric nuclei in 2D images have traditionally been manual. This is exemplified by attempts to measure chromosome position in sperm samples, typically by dividing the nucleus into zones, and manually scoring which zone a fluorescence in-situ hybridisation (FISH) signal lies in. This is time consuming, limiting the number of nuclei that can be analyzed, and prone to subjectivity. We have developed a new approach for automated mapping of FISH signals in asymmetric nuclei, integrated into an existing image analysis tool for nuclear morphology. Automatic landmark detection defines equivalent structural regions in each nucleus, then dynamic warping of the FISH images to a common shape allows us to generate a composite of the signal within the entire cell population. Using this approach, we mapped the positions of the sex chromosomes and two autosomes in three mouse lineages ( Mus musculus domesticus , Mus musculus musculus and Mus spretus ). We found that in all three, chromosomes 11 and 19 tend to interact with each other, but are shielded from interactions with the sex chromosomes. This organization is conserved across 2 million years of mouse evolution., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Current methods for chromosome painting via fluorescence in situ hybridization (FISH) are costly, time-consuming, and limited in complexity. In contrast to conventional sources of probe, Oligopaints are computationally designed, synthesized on microarrays, and amplified by PCR. This approach allows for precise control over the sequences they target, which can range from a few kilobases to entire chromosomes with the same basic protocol. We have utilized the flexibility and scalability of Oligopaints to generate low-cost and renewable chromosome paints for Drosophila, mouse, and human chromosomes. These Oligopaint libraries can be customized to label any genomic feature(s) in a chromosome-wide manner. Additionally, this method is compatible with sequential FISH to label entire genomes with a single denaturation step. Here, we outline a protocol and considerations to scale the Oligopaint technology for fluorescent labeling of whole chromosomes.

Analysis of chromosome pairing has been an important tool to assess the genetic similarity of homologous and homoeologous chromosomes in polyploids. However, it is technically challenging to monitor the pairing of specific chromosomes in polyploid species, especially for plant species with a large number of small chromosomes. We developed oligonucleotide-based painting probes for four different potato chromosomes. We demonstrate that these probes are robust enough to monitor a single chromosome throughout the prophase I of meiosis in polyploid Solanum species. Cultivated potato (Solanum tuberosum, 2n = 4x = 48) is an autotetraploid. We demonstrate that the four copies of each potato chromosome pair as a quadrivalent in 66-78% of the meiotic cells at the pachytene stage. Solanum demissum (2n = 6x = 72) is a hexaploid and has been controversial regarding its nature as an autopolyploid or allopolyploid. Interestingly, no hexavalent pairing was observed in meiosis. Instead, we observed three independent bivalents in 83-98% of the meiotic cells at late diakinesis and early metaphase I for the four chromosomes. These results suggest that S. demissum has evolved into a cytologically stable state with predominantly bivalent pairing in meiosis.

Dioecious species accounted for 6% of all plant species, including a number of crops and economically important species, such as poplar. However, sex determination and sex chromosome evolution have been studied only in few dioecious species. In poplar, the sex-determining locus was mapped to chromosome 19. Interestingly, this locus was mapped to either a peritelomeric or a centromeric region among different poplar species. We developed an oligonucleotide (oligo)-based chromosome painting probe based on the sequence of chromosome 19 from Populus trichocarpa. We performed chromosome painting in P. tomentosa and P. deltoides. Surprisingly, the distal end on the short arm of chromosome 19, which corresponds to the location of the sex-determining locus reported in several species, was not painted in both species. Thus, the DNA sequences associated with this region have not been anchored to the current chromosome 19 pseudomolecule, which was confirmed by painting of somatic metaphase chromosome 19 of P. trichocarpa. Interestingly, the unpainted distal ends of the two chromosome 19 did not pair at the pachytene stage in 22-24% of the meiotic cells in the two species, suggest that these regions from the sex chromosomes have structurally diverged from each other, resulting in the reduced pairing frequency. These results shed light on divergence of a pair of young sex chromosomes in poplar.

Pigeons and doves (Columbiformes) are one of the oldest and most diverse extant lineages of birds. However, the karyotype evolution within Columbiformes remains unclear. To delineate the synteny-conserved segments and karyotypic differences among four Columbidae species, we used chromosome painting from Gallus gallus (GGA, 2n = 78) and Leucopternis albicollis (LAL, 2n = 68). Besides that, a set of painting probes for the eared dove, Zenaida auriculata (ZAU, 2n = 76), was generated from flow-sorted chromosomes. Chromosome painting with GGA and ZAU probes showed conservation of the first ten ancestral pairs in Z. auriculata, Columba livia, and Columbina picui, while in Leptotila verreauxi, fusion of the ancestral chromosomes 6 and 7 was observed. However, LAL probes revealed a complex reorganization of ancestral chromosome 1, involving paracentric and pericentric inversions. Because of the presence of similar intrachromosomal rearrangements in the chromosomes corresponding to GGA1q in the Columbidae and Passeriformes species but without a common origin, these results are consistent with the recent proposal of divergence within Neoaves (Passerea and Columbea). In addition, inversions in chromosome 2 were identified in C. picui and L. verreauxi. Thus, in four species of distinct genera of the Columbidae family, unique chromosomal rearrangements have occurred during karyotype evolution, confirming that despite conservation of the ancestral syntenic groups, these chromosomes have been modified by the occurrence of intrachromosomal rearrangements.

Glyphorynchus spirurus (GSP), also called the Wedge-billed Woodcreeper (Furnariidae) has an extensive distribution in the Americas, including the Atlantic coast of Brazil. Nevertheless, there is no information about its karyotype or genome organization. To contribute to the knowledge of chromosomal evolution in Passeriformes we analysed the karyotype of Glyphorynchus spirurus by classic and molecular cytogenetics methods. We show that Glyphorynchus spirurus has a 2n = 80 karyotype with a fundamental number (FN) of 84, similar to the avian putative ancestral karyotype (PAK). Glyphorynchus spirurus pair 1 was heteromorphic in the Tapajós population whereby the short arms varied in sizes, possibly due to a pericentric inversion, as described in other Furnariidae birds. FISH with the Histone H5 probe revealed a signal in the pericentromeric region of G. spirurus chromosome 5 and rDNA 18S showed interstitial signal in GSP-1. Chromosome painting with Gallus gallus (GGA) macrochromosomes 1-9 probes showed disruption of chromosome syntenies of GGA-1, 2 and 4 by fission in Glyphorynchus spirurus. Our results confirm that the GGA1 centric fission is a synapomorphic character for the phylogenetic branch composed of Strigiformes, Passeriformes, Columbiformes and Falconiformes. On the other hand, the GGA-2 fission is reported here for the first time in Passeriformes. Chromosome painting with BOE whole chromosome probes confirmed these rearrangements in Glyphorynchus spirurus revealed by Gallus gallus 1-9 probes, in addition to enabling the establishment of genome-wide homology map., Competing Interests: The authors have declared that no competing interests exist.

Competing Interests: The authors have declared that no competing interests exist.

Background: The chromosome-specific probe is a fundamental tool of chromosome painting and has been commonly applied in mammalian species. The technology, however, has not been widely applied in plants due to a lack of methodologies for probe development. Identification and labeling of a large number of oligonucleotides (oligos) specific to a single chromosome offers us an opportunity to establish chromosome-specific probes in plants. However, never before has whole chromosome painting been performed in rice., Results: We developed a pooled chromosome 9-specific probe in rice, which contains 25,000 oligos based on the genome sequence of a japonica rice (Oryza sativa L., AA, 2n = 2× = 24). Chromosome 9 was easily identified in both japonica and indica rice using this chromosome 9-painting probe. The probe was also successfully used to identify and characterize chromosome 9 in additional lines of O. sativa, a translocation line, two new aneuploids associated with chromosome 9 and a wild rice (Oryza eichingeri A. Peter, CC, 2n = 2× = 24)., Conclusion: The study reveals that a pool of oligos specific to a chromosome is a useful tool for chromosome painting in rice.

Background: The family Phyllostomidae (Chiroptera) shows wide morphological, molecular and cytogenetic variation; many disagreements regarding its phylogeny and taxonomy remains to be resolved. In this study, we use chromosome painting with whole chromosome probes from the Phyllostomidae Phyllostomus hastatus and Carollia brevicauda to determine the rearrangements among several genera of the Nullicauda group (subfamilies Gliphonycterinae, Carolliinae, Rhinophyllinae and Stenodermatinae)., Results: These data, when compared with previously published chromosome homology maps, allow the construction of a phylogeny comparable to those previously obtained by morphological and molecular analysis. Our phylogeny is largely in agreement with that proposed with molecular data, both on relationships between the subfamilies and among genera; it confirms, for instance, that Carollia and Rhinophylla, previously considered as part of the same subfamily are, in fact, distant genera., Conclusions: The occurrence of the karyotype considered ancestral for this family in several different branches suggests that the diversification of Phyllostomidae into many subfamilies has occurred in a short period of time. Finally, the comparison with published maps using human whole chromosome probes allows us to track some syntenic associations prior to the emergence of this family.

Deletions in the 4p16.3 region are associated with Wolf-Hirschhorn syndrome (WHS), a contiguous gene deletion syndrome involving variable size deletions. In this study, we perform a cytogenomic integrative analysis combining classical cytogenetic methods, fluorescence in situ hybridization (FISH), chromosomal microarray analysis (CMA), and systems biology strategies, to establish the cytogenomic profile involving the 4p16.3 critical region and suggest WHS-related intracellular cell signaling cascades. The cytogenetic and clinical patient profiles were evaluated. We characterized 12 terminal deletions, one interstitial deletion, two ring chromosomes, and one classical translocation 4;8. CMA allowed delineation of the deletions, which ranged from 3.7 to 25.6 Mb with breakpoints from 4p16.3 to 4p15.33. Furthermore, the smallest region of overlapping (SRO) encompassed seven genes in a terminal region of 330 kb in the 4p16.3 region, suggesting a region of susceptibility to convulsions and microcephaly. Therefore, molecular interaction networks and topological analysis were performed to understand these WHS-related symptoms. Our results suggest that specific cell signaling pathways including dopamine receptor, NAD+ nucleosidase activity, and fibroblast growth factor-activated receptor activity are associated with the diverse pathological WHS phenotypes and their symptoms. Additionally, we identified 29 hub-bottlenecks (H-B) nodes with a major role in WHS.

Brachypodium distachyon provides a particularly appealing object for molecular cytogenetic analysis due to its compact genome and low repetitive DNA content, as well as low (x = 5) basic number of chromosomes easily identifiable on the basis of their morphometric features. Some of these features, such as genome compactness, are shared by the other members of the genus, thus making them amenable for comparative cytogenetic mapping. Cytogenetic infrastructure established for B. distachyon was initially based on fluorescence in situ hybridization with various tandemly repeated sequences as probes. The molecular cytogenetic studies advanced greatly with the development of B. distachyon large DNA insert genomic libraries. These resources coupled with the access to the fully sequenced genome of B. distachyon enabled chromosome painting in monocots for the first time. This pioneering work was subsequently extended to other Brachypodium species, allowing insight into grass karyotype evolution. In this protocol we describe the methods of making somatic and meiotic chromosome preparations, probe labeling, FISH with BAC clones, a strategy for chromosome barcoding and chromosome painting in B. distachyon, and comparative chromosome painting in the other Brachypodium species.

In comparison with general FISH for preparing probes in terms of time and cost, synthesized oligonucleotide (oligo hereafter) probes for FISH have many advantages such as ease of design, synthesis, and labeling. Low cost and high sensitivity and resolution of oligo probes greatly simplify the FISH procedure as a simple, fast, and efficient method of chromosome identification. In this study, we developed new oligo and oligo multiplex probes to accurately and efficiently distinguish wheat (Triticum aestivum, 2n = 6x, AABBDD) and Thinopyrum bessarabicum (2n = 2x = 14, JJ) chromosomes. The oligo probes contained more nucleotides or more repeat units that produced stronger signals for more efficient chromosome painting. Four Th. bessarabicum-specific oligo probes were developed based on genomic DNA sequences of Th. bessarabicum chromosome arm 4JL, and one of them (oligo DP4J27982) was pooled with the oligo multiplex #1 to simultaneously detect wheat and Th. bessarabicum chromosomes for quick and accurate identification of Chinese Spring (CS) - Th. bessarabicum alien chromosome introgression lines. Oligo multiplex #4 revealed chromosome variations among CS and eight wheat cultivars by a single round of FISH analysis. This research demonstrated the high efficiency of using oligos and oligo multiplexes in chromosome identification and manipulation.

Sokolov's dwarf hamster (Cricetulus sokolovi) is the least studied representative of the striped hamsters (Cricetulus barabensis species group), the taxonomy of which remains controversial. The species was described based on chromosome morphology, but neither the details of the karyotype nor the phylogenetic relationships with other Cricetulus are known. In the present study, the karyotype of C. sokolovi was examined using cross-species chromosome painting. Molecular and cytogenetic data were employed to determine the phylogenetic position of Sokolov's hamster and to analyze the potential pathways of chromosome evolution in Cricetulus. Both the chromosome and molecular data support the species status of Sokolov's hamster. Phylogenetic analysis of the CYTB data placed C. sokolovi as sister to all other striped hamsters (sequence divergence of 8.1%). FISH data revealed that the karyotype of C. sokolovi is highly rearranged, with the most parsimonious scenario of its origin implying at least 4 robertsonian events and a centromere shift. Comparative cytogenetic data on Cricetinae suggest that their evolutionary history includes both periods of chromosomal conservatism and episodes of rapid chromosomal change., (© 2017 S. Karger AG, Basel.)

Eukaryote genomes are frequently burdened with the presence of supernumerary (B) chromosomes. Their origin is frequently investigated by chromosome painting, under the hypothesis that sharing the repetitive DNA sequences contained in the painting probes is a sign of common descent. However, the intragenomic mobility of many anonymous DNA sequences contained in these probes (e.g., transposable elements) adds high uncertainty to this conclusion. Here we test the validity of chromosome painting to investigate B chromosome origin by comparing its results for seven B chromosome types in two fish species genus Astyanax, with those obtained (1) by means of the physical mapping of 18S ribosomal DNA (rDNA), H1 histone genes, the As51 satellite DNA and the (AC)15 microsatellite, and (2) by comparing the nucleotide sequence of one of these families (ITS regions from ribosomal DNA) between genomic DNA from B-lacking individuals in both species and the microdissected DNA from two metacentric B chromosomes found in these same species. Intra- and inter-specific painting suggested that all B chromosomes that were assayed shared homologous DNA sequences among them, as well as with a variable number of A chromosomes in each species. This finding would be consistent with a common origin for all seven B chromosomes analyzed. By contrast, the physical mapping of repetitive DNA sequences failed to give support to this hypothesis, as no more than two B-types shared a given repetitive DNA. Finally, sequence analysis of the ITS regions suggested that at least some of the B chromosomes could have had a common origin.

Fluorescence in situ hybridization (FISH) is a molecular cytogenetic technique. It identifies the location of DNA loci and RNAs, including nascent RNAs in the process of being transcribed, within individual cells. Great advances in fluorescent dye technology and technique sensitivity, combined with developments in light microscopy and imaging software have made it widely accessible and have expanded the range of applications in basic research as well as in diagnostics. Being able to perform RNA hybridization, DNA hybridization, and protein immunofluorescence consecutively on the same sample is an invaluable tool to study RNA expression in relation to their gene loci and to map RNA and DNA in relation to nuclear or cellular structures. This has contributed to enormous progress in understanding basal mechanisms of male and female meiosis in different animal model systems. In this chapter we describe in detail the protocols for FISH based techniques applied to study gene expression dynamics and nuclear architecture of chicken oocytes during meiotic prophase I. These techniques can be easily performed in any molecular and cell biology laboratory and be adapted to different systems and to different phases of gametogenesis.

Pteropodidae and Hipposideridae are 2 of the 9 chiropteran families that occur on Madagascar. Despite major advancements in the systematic study of the island's bat fauna, few karyotypic data exist for endemic species. We utilized G- and C-banding in combination with chromosome painting with Myotismyotis probes to establish a genome-wide homology among Malagasy species belonging to the families Pteropodidae (Pteropus rufus 2n = 38; Rousettus madagascariensis, 2n = 36), Hipposideridae (Hipposideros commersoni s.s., 2n = 52), and a single South African representative of the Rhinolophidae (Rhinolophus clivosus, 2n = 58). Painting probes of M. myotis detected 26, 28, 28, and 29 regions of homology in R. madagascariensis, P. rufus, H. commersoni s.s, and R. clivosus, respectively. Translocations, pericentric inversions, and heterochromatin additions were responsible for karyotypic differences amongst the Malagasy pteropodids. Comparative chromosome painting revealed a novel pericentric inversion on P. rufus chromosome 4. Chromosomal characters suggest a close evolutionary relationship between Rousettus and Pteropus. H. commersoni s.s. shared several chromosomal characters with extralimital congeners but did not exhibit 2 chromosomal synapomorphies proposed for Hipposideridae. This study provides further insight into the ancestral karyotypes of pteropodid and hipposiderid bats and corroborates certain molecular phylogenetic hypotheses., (© 2016 S. Karger AG, Basel.)

Background: Previous cross-species painting studies with probes from chicken (Gallus gallus) chromosomes 1-10 and a paint pool of nineteen microchromosomes have revealed that the drastic karyotypic reorganization in Accipitridae is due to extensive synteny disruptions and associations. However, the number of synteny association events and identities of microchromosomes involved in such synteny associations remain undefined, due to the lack of paint probes derived from individual chicken microchromosomes. Moreover, no genome-wide homology map between Accipitridae species and other avian species with atypical karyotype organization has been reported till now, and the karyotype evolution within Accipitriformes remains unclear., Results: To delineate the synteny-conserved segments in Accipitridae, a set of painting probes for the griffon vulture, Gyps fulvus (2n = 66) was generated from flow-sorted chromosomes. Together with previous generated probes from the stone curlew, Burhinus oedicnemus (2n = 42), a Charadriiformes species with atypical karyotype organization, we conducted multidirectional chromosome painting, including reciprocal chromosome painting between B. oedicnemus and G. fulvus and cross-species chromosome painting between B. oedicnemus and two accipitrid species (the Himalayan griffon, G. himalayensis 2n = 66, and the common buzzard, Buteo buteo, 2n = 68). In doing so, genome-wide homology maps between B. oedicnemus and three Accipitridae species were established. From there, a cladistic analysis using chromosomal characters and mapping of chromosomal changes on a consensus molecular phylogeny were conducted in order to search for cytogenetic signatures for different lineages within Accipitriformes., Conclusion: Our study confirmed that the genomes of the diurnal birds of prey, especially the genomes of species in Accipitriformes excluding Cathartidae, have been extensively reshuffled when compared to other bird lineages. The chromosomal rearrangements involved include both fusions and fissions. Our chromosome painting data indicated that the Palearctic common buzzard (BBU) shared several common chromosomal rearrangements with some Old World vultures, and was found to be more closely related to other Accipitridae than to Neotropical buteonine raptors from the karyotypic perspective. Using both a chromosome-based cladistic analysis as well as by mapping of chromosomal differences onto a molecular-based phylogenetic tree, we revealed a number of potential cytogenetic signatures that support the clade of Pandionidae (PHA) + Accipitridae. In addition, our cladistic analysis using chromosomal characters appears to support the placement of osprey (PHA) in Accipitridae.

Comparative chromosome painting is a powerful tool to study the evolution of chromosomes and genomes. Analyzing karyotype evolution in cruciferous plants highlights the origin of aberrant chromosomes in apomictic Boechera and further establishes the cruciferous plants as important model system for our understanding of plant chromosome and genome evolution., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Chromosome-specific painting is a powerful technique in molecular cytogenetic and genome research. We developed an oligonucleotide (oligo)-based chromosome painting technique in cucumber (Cucumis sativus) that will be applicable in any plant species with a sequenced genome. Oligos specific to a single chromosome of cucumber were identified using a newly developed bioinformatic pipeline and then massively synthesized de novo in parallel. The synthesized oligos were amplified and labeled with biotin or digoxigenin for use in fluorescence in situ hybridization (FISH). We developed three different probes with each containing 23,000-27,000 oligos. These probes spanned 8.3-17 Mb of DNA on targeted cucumber chromosomes and had the densities of 1.5-3.2 oligos per kilobases. These probes produced FISH signals on a single cucumber chromosome and were used to paint homeologous chromosomes in other Cucumis species diverged from cucumber for up to 12 million years. The bulked oligo probes allowed us to track a single chromosome in early stages during meiosis. We were able to precisely map the pairing between cucumber chromosome 7 and chromosome 1 of Cucumis hystrix in a F1 hybrid. These two homeologous chromosomes paired in 71% of prophase I cells but only 25% of metaphase I cells, which may provide an explanation of the higher recombination rates compared to the chiasma frequencies between homeologous chromosomes reported in plant hybrids., (Copyright © 2015 by the Genetics Society of America.)

The domesticated guinea pig, Cavia porcellus (Hystricomorpha, Rodentia), is an important laboratory species and a model for a number of human diseases. Nevertheless, genomic tools for this species are lacking; even its karyotype is poorly characterized. The guinea pig belongs to Hystricomorpha, a widespread and important group of rodents; so far the chromosomes of guinea pigs have not been compared with that of other hystricomorph species or with any other mammals. We generated full sets of chromosome-specific painting probes for the guinea pig by flow sorting and microdissection, and for the first time, mapped the chromosomal homologies between guinea pig and human by reciprocal chromosome painting. Our data demonstrate that the guinea pig karyotype has undergone extensive rearrangements: 78 synteny-conserved human autosomal segments were delimited in the guinea pig genome. The high rate of genome evolution in the guinea pig may explain why the HSA7/16 and HSA16/19 associations presumed ancestral for eutherians and the three syntenic associations (HSA1/10, 3/19, and 9/11) considered ancestral for rodents were not found in C. porcellus. The comparative chromosome map presented here is a starting point for further development of physical and genetic maps of the guinea pig as well as an aid for genome assembly assignment to specific chromosomes. Furthermore, the comparative mapping will allow a transfer of gene map data from other species. The probes developed here provide a genomic toolkit, which will make the guinea pig a key species to unravel the evolutionary biology of the Hystricomorph rodents.

This review describes my personal reflections on the development of whole chromosome painting using fluorescence in situ hybridization and how my laboratory applied the technology in humans and in animal models. The trials and triumphs of the early years are emphasized, along with some of the scientific surprises that were encountered along the way. Scientific issues that my laboratory addressed using chromosome painting technologies are summarized and related to questions in radiation dosimetry, chemical clastogenesis, translocation persistence, and translocation frequencies in unexposed people. A description is provided of scientific controversies that were encountered and how they were resolved. I hope this paper will encourage young scientists to follow their passions and pursue their scientific dreams even if the task seems daunting and the circumstances appear exceedingly difficult. In my case the journey has been challenging, exciting, and richly rewarding on many levels., (Copyright © 2014 Elsevier B.V. All rights reserved.)

To determine the appropriateness of chromosome painting for identifying genomic elements in rye, we microdissected the 1R and 1RS chromosomes from rye (Secale cereale L. var. King II) and wheat-rye addition line 1RS, respectively. Degenerate oligonucleotide primed - polymerase chain reaction (DOP-PCR) amplification of 1R and 1RS products from dissected chromosomes were used as probes to hybridize to metaphase chromosomes of rye, wheat-rye addition lines 1R and 1RS, translocation line 1RS.1BL, and allohexaploid triticale. The results showed that (i) the hybridization signal distribution patterns on rye chromosomes using 1R-derived DOP-PCR products as the probe were similar to those using 1RS-derived DOP-PCR products as the probe; (ii) 1R and (or) 1RS could not be distinguished from other rye chromosomes solely by the hybridization patterns using 1R- and (or) 1RS-derived DOP-PCR products as the probe; (iii) rye chromosomes and (or) rye chromosome fragments could be clearly identified in wheat-rye hybrids using either 1R- or 1RS-derived DOP-PCR products as the probe and could be more accurate in the nontelomeric region than using genomic in situ hybridization (GISH). Our results suggested that 1R- and (or) 1RS-derived DOP-PCR products contain many repetitive DNA sequences, are similar on different rye chromosomes, are R-genome specific, and can be used to identify rye chromosomes and chromosome fragments in wheat-rye hybrids. Our research widens the application range of chromosome painting in plants.

Background: Pronghorn (Antilocapridae, 2n = 58) and saola (Bovidae, 2n = 50) are members of Pecora, a highly diversified group of even-toed hoofed mammals. Karyotypes of these species were not involved in chromosome painting studies despite their intriguing phylogenetic positions in Pecora., Results: To trace the chromosome evolution during very fast radiation of main families from the common Pecoran ancestor, high-resolution comparative chromosome maps of pronghorn and saola with human (HSA) and dromedary camel (CDR) painting probes were established. The human and dromedary camel painting probes revealed 50 and 64 conserved segments respectively in the pronghorn genome, while 51 and 63 conserved segments respectively in the saola genome. Integrative analysis with published comparative maps showed that inversions in chromosomes homologous to CDR19/35/19 (HSA 10/20/10), CDR12/34/12 (HSA12/22/12/22), CDR10/33/10 (HSA 11) are present in representatives of all five living Pecoran families. The pronghorn karyotype could have formed from a putative 2n = 58 Pecoran ancestral karyotype by one fission and one fusion and that the saola karyotype differs from the presumed 2n = 60 bovid ancestral karyotype (2n = 60) by five fusions., Conclusion: The establishment of high-resolution comparative maps for pronghorn and saola has shed some new insights into the putative ancestral karyotype, chromosomal evolution and phylogenic relationships in Pecora. No cytogenetic signature rearrangements were found that could unite the Antilocapridae with Giraffidae or with any other Pecoran families. Our data on the saola support a separate position of Pseudorigyna subtribe rather than its affinity to either Bovina or Bubalina, but the saola phylogenetic position within Bovidae remains unresolved.

Supernumerary (B) chromosomes have been shown to contain a wide variety of repetitive sequences. For this reason, fluorescent in situ hybridisation (FISH) is a useful tool for ascertaining the origin of these genomic elements, especially when combined with painting from microdissected B chromosomes. In order to investigate the origin of B chromosomes in the fish species Astyanax paranae, these two approaches were used along with PCR amplification of specific DNA sequences obtained from the B chromosomes and its comparison with those residing in the A chromosomes. Remarkably, chromosome painting with the one-arm metacentric B chromosome probe showed hybridization signals on entire B chromosome, while FISH mapping revealed the presence of H1 histone and 18S rDNA genes symmetrically placed in both arms of the B chromosome. These results support the hypothesis that the B chromosome of A. paranae is an isochromosome. Additionally, the chromosome pairs Nos. 2 or 23 are considered the possible B chromosome ancestors since both contain syntenic H1 and 18S rRNA sequences. The analysis of DNA sequence fragments of the histone and rRNA genes obtained from the microdissected B chromosomes showed high similarity with those obtained from 0B individuals, which supports the intraspecific origin of B chromosomes in A. paranae. Finally, the population hereby analysed showed a female-biased B chromosome presence suggesting that B chromosomes in this species could influence sex determinism.

Chromosome painting based on fluorescence in situ hybridization (FISH) has played an important role in chromosome identification and research into chromosome rearrangements, diagnosis of chromosome abnormalities and evolution in human and animal species. However, it has not been applied widely in plants due to the large amounts of dispersed repetitive sequences in chromosomes. In the present work, a chromosome painting method for single-copy gene pools in Cucumis sativus was successfully developed. Gene probes with sizes above 2 kb were detected consistently. A cucumber karyotype was constructed based on FISH using a cocktail containing chromosome-specific gene probes. This single-copy gene-based chromosome painting (ScgCP) technique was performed by PCR amplification, purification, pooling, labeling and hybridization onto chromosome spreads. Gene pools containing sequential genes with an interval less than 300 kb yielded painting patterns on pachytene chromosomes. Seven gene pools corresponding to individual chromosomes unambiguously painted each chromosome pair of C. sativus. Three mis-aligned regions on chromosome 4 were identified by the painting patterns. A probe pool comprising 133 genes covering the 8 Mb distal end of chromosome 4 was used to evaluate the potential utility of the ScgCP technique for chromosome rearrangement research through cross-species FISH in the Cucumis genus. Distinct painting patterns of this region were observed in C. sativus, C. melo and C. metuliferus species. A comparative chromosome map of this region was constructed between cucumber and melon. With increasing sequence resources, this ScgCP technique may be applied on any other sequenced species for chromosome painting research., (© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.)