Chromosome-specific Painting Probes Research Articles

High rates of structural rearrangements have shaped the chromosome evolution in dysploid Phaseolus beans.

Karyotypes evolve through numerical and structural chromosome rearrangements. We show that Phaseolus leptostachyus, a wild bean, underwent a rapid genome reshuffling associated with the reduction from 11 to 10 chromosome pairs, but without whole genome duplication, the highest chromosome evolution rate known for plants. Plant karyotypes evolve through structural rearrangements often associated with polyploidy or dysploidy. The genus Phaseolus comprises ~ 90 species, five of them domesticated due to their nutritional relevance. Most of the species have 2n = 22 karyotypes and are highly syntenic, except for three dysploid karyotypes of species from the Leptostachyus group (2n = 20) that have accumulated several rearrangements. Here, we investigated the degrees of structural rearrangements among Leptostachyus and other Phaseolus groups by estimating their chromosomal evolution rates (CER). For this, we combined our oligo-FISH barcode system for beans and chromosome-specific painting probes for chromosomes 2 and 3, with rDNA and a centromeric probe to establish chromosome orthologies and identify structural rearrangements across nine Phaseolus species. We also integrated the detected rearrangements with a phylogenomic approach to estimate the CERs for each Phaseolus lineage. Our data allowed us to identify translocations, inversions, duplications and deletions, mostly in species belonging to the Leptostachyus group. Phaseolus leptostachyus showed the highest CER (12.31 rearrangements/My), a tenfold increase in contrast to the 2n = 22 species analysed. This is the highest rate known yet for plants, making it a model species for investigating the mechanisms behind rapid genome reshuffling in early species diversification.

Chromosome-specific painting in Thinopyrum species using bulked oligonucleotides.

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, EbEb) has a close genetic relationship with diploid Th. elongatum (EeEe), with five chromosomes (1E, 2E, 3E, 6E, and 7E) maintaining complete synteny in the two species except for a reciprocal translocation between 4 and 5Eb. 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.

Oligo-FISH of Populus simonii Pachytene Chromosomes Improves Karyotyping and Genome Assembly.

Poplar was one of the first woody species whose individual chromosomes could be identified using chromosome specific painting probes. Nevertheless, high-resolution karyotype construction remains a challenge. Here, we developed a karyotype based on the meiotic pachytene chromosome of Populus simonii which is a Chinese native species with many excellent traits. This karyotype was anchored by oligonucleotide (oligo)-based chromosome specific painting probes, a centromere-specific repeat (Ps34), ribosomal DNA, and telomeric DNA. We updated the known karyotype formula for P. simonii to 2n = 2x = 38 = 26m + 8st + 4t and the karyotype was 2C. The fluorescence in situ hybridization (FISH) results revealed some errors in the current P. simonii genome assembly. The 45S rDNA loci were located at the end of the short arm of chromosomes 8 and 14 by FISH. However, they were assembled on pseudochromosomes 8 and 15. In addition, the Ps34 loci were distributed in every centromere of the P. simonii chromosome in the FISH results, but they were only found to be present in pseudochromosomes 1, 3, 6, 10, 16, 17, 18, and 19. Our results reveal that pachytene chromosomes oligo-FISH is a powerful tool for constructing high-resolution karyotypes and improving the quality of genome assembly.

Comparative chromosome maps between the stone curlew and three ciconiiform species (the grey heron, little egret and crested ibis)

BackgroundPrevious cytogenetic studies show that the karyotypes of species in Ciconiiformes vary considerably, from 2n = 52 to 78. Their karyotypes include different numbers of small to minute bi-armed chromosomes that have evolved probably by fusions of two ancestral microchromosomes, besides macrochromosomes and dot-like microchromosomes. However, it is impossible to define the inter-species homologies of such small-sized bi-armed chromosomes based on chromosome morphology and banding characteristics. Although painting probes from the chicken (Gallus gallus, GGA) chromosomes 1–9 and Z have been widely used to investigate avian chromosome homologies, GGA microchromosome probes are rarely used in these studies because most GGA microchromosome probes generated by flow sorting often contain multiple GGA microchromosomes. In contrast, the stone curlew (Burhinus oedicnemus, BOE, Charadriiformes) has an atypical low diploid chromosome number (42) karyotype and only 4 pairs of dot-like microchromosomes; a set of chromosome-specific painting probes that cover all BOE chromosomes has been generated. To get a genome-wide view of evolutionary chromosomal rearrangements in different lineages of Ciconiiformes, we used BOE painting probes instead of GGA painting probes to analyze the karyotypes of three ciconiiform species belonging to two different families: the eastern grey heron (Ardea cinerea, ACI, 2n = 64, Ardeidae), the little egret (Egretta garzetta, EGA, 2n = 64, Ardeidae) and the crested ibis (Nipponia nippon, NNI, 2n = 68, Threskiornithidae).ResultsBOE painting probes display the same hybridization pattern on chromosomes of ACI and EGA, while a different hybridization pattern is observed on chromosomes of NNI. BOE autosome probes detected 21 conserved homologous segments and 5 fusions on the sixteen pairs of recognizable chromosomes of ACI and EGA, while 16 conserved homologous segments and 4 fusions were found on the twelve pairs of recognizable chromosomes of NNI. Only a portion of smaller bi-armed chromosomes in the karyotypes of the ciconiiform species could have evolved from fusions of ancestral microchromosomes. In particular BOE 5, which is the result of a fusion between two segments homologous to GGA 7 and 8 respectively, was retained also as either a single chromosome in ACI (ACI 5) and EGA (EGA 5) or had fused with a part of the BOE 10 equivalent in NNI (NNI 5).ConclusionOur painting results indicate that different chromosome rearrangements occur in different ciconiiform lineages. Some of the small-sized bi-armed chromosomes in ACI, EGA and NNI are derived from the fusions of two microchromosomes, indicating that microchromosome fusions play an important role in ciconiiform chromosome evolution. The fusion segment homologous to GGA 7 and 8 is a potential cytogenetic signature that unites Ardeidae and Threskiornithidae.

Chromosome Painting Based on Bulked Oligonucleotides in Cotton.

Chromosome painting is one of the key technologies in cytogenetic research, which can accurately identify chromosomes or chromosome regions. Oligonucleotide (oligo) probes designed based on genome sequences have both flexibility and specificity, which would be ideal probes for fluorescence in situ hybridization (FISH) analysis of genome structure. In this study, the bulked oligos of the two arms of chromosome seven of cotton were developed based on the genome sequence of Gossypium raimondii (DD, 2n = 2× = 26), and each arm contains 12,544 oligos. Chromosome seven was easily identified in both D genome and AD genome cotton species using the bulked chromosome-specific painting probes. Together with 45S ribosomal DNA (rDNA) probe, the chromosome-specific painting probe was also successfully used to correct the chromosomal localization of 45S rDNA in G. raimondii. The study reveals that bulked oligos specific to a chromosome is a useful tool for chromosome painting in cotton.

Micronucleus formation in human cancer cells is biased by chromosome size.

Chromosomal instability is one of the hallmarks of cancer and caused by chromosome missegregation during mitosis, a process frequently associated with micronucleus formation. Micronuclei are formed when chromosomes fail to join a daughter nucleus during cell division and are surrounded by their own nuclear membrane. Although it has been commonly assumed that the gain or loss of specific chromosomes is random during compromised cell division, recent data suggest that the size of chromosomes can impact on chromosome segregation fidelity. To test whether chromosome missegregation rates scale with chromosome size in primary human cancer cells, we assessed chromosome sequestration into micronuclei in patient-derived primary NCH149 glioblastoma cells, which display high-level numerical chromosome instability (CIN), pronounced spontaneous micronucleus formation but virtually no structural CIN. The cells were analyzed by interphase fluorescence in situ hybridization using chromosome-specific painting probes for all chromosomes. Overall, 33% of early passage NCH149 cells harbored micronuclei. Entrapment within a micronucleus clearly correlated with chromosome size with larger chromosomes being significantly more frequently missegregated into micronuclei than smaller chromosomes in primary glioblastoma cells. These findings extend the concept that chromosome size determines segregation fidelity by implying that size-specific micronucleus entrapment occurs in primary human cancer cells as well.

Isolating Chromosomes of the Komodo Dragon: New Tools for Comparative Mapping and Sequence Assembly

We developed new tools to build a high-quality chromosomal map of the Komodo dragon (Varanus komodoensis) available for cross-species phylogenomic analyses. First, we isolated chromosomes by flow sorting and determined the chromosome content of each flow karyotype peak by FISH. We then isolated additional Komodo dragon chromosomes by microdissection and amplified chromosome-specific DNA pools. The chromosome-specific DNA pools can be sequenced, assembled, and mapped by next-generation sequencing technology. The chromosome-specific paint probes can be used to investigate karyotype evolution through cross-species chromosome painting. Overall, the set of chromosome-specific DNA pools of V. komodoensis provides new tools for detailed phylogenomic analyses of Varanidae and squamates in general.

Pooling strategy and chromosome painting characterize a living zebroid for the first time.

We have investigated the complex karyotype of a living zebra-donkey hybrid for the first time using chromosome-specific painting probes produced from flow-sorted chromosomes from a zebra (Equus burchelli) and horse (Equus caballus). As the chromosomes proved difficult to distinguish from one another, a successful new strategy was devised to resolve the difficulty and characterize each chromosome. This was based on selecting five panels of whole chromosome painting probes that could differentiate zebra and donkey chromosomes by labelling the probes with either FITC or Cy3 fluorochromes. Each panel was hybridized sequentially to the same G-Q-banded metaphases and the results combined so that every zebra and donkey chromosome in each suitable metaphase could be identified. A diploid number of 2n = 53, XY was found, containing haploid sets of 22 chromosomes from the zebra and 31 chromosomes from the donkey, without evidence of chromosome rearrangement. This new strategy, developed for the first time, may have several applications in the resolution of other complex hybrid karyotypes and chromosomal aberrations.

Chromosomal Aberrations in Large Japanese Field Mice (Apodemus speciosus) Captured near Fukushima Dai-ichi Nuclear Power Plant.

Since the Fukushima Dai-ichi Nuclear Power Plant accident, radiation effects on nonhuman biota in the contaminated areas have been a major concern. Here, we analyzed the frequencies of chromosomal aberrations (translocations and dicentrics) in the splenic lymphocytes of large Japanese field mice (Apodemus speciosus) inhabiting Fukushima Prefecture. A. speciosus chromosomes 1, 2, and 5 were flow-sorted in order to develop A. speciosus chromosome-specific painting probes, and FISH (fluorescence in situ hybridization) was performed using these painting probes to detect the translocations and dicentrics. The average frequency of the translocations and dicentrics per cell in the heavily contaminated area was significantly higher than the frequencies in the case of the noncontaminated control area and the slightly and moderately contaminated areas, and this aberration frequency in individual mice tended to roughly increase with the estimated dose rates and accumulated doses. In all four sampling areas, the proportion of aberrations occurring in chromosome 2 was approximately >3 times higher than that in chromosomes 1 and 5, which suggests that A. speciosus chromosome 2 harbors a fragile site that is highly sensitive to chromosome breaks induced by cellular stress such as DNA replication. The elevated frequency of chromosomal aberrations in A. speciosus potentially resulting from the presence of a fragile site in chromosome 2 might make it challenging to observe the mild effect of chronic low-dose-rate irradiation on the induction of chromosomal aberrations in A. speciosus inhabiting the contaminated areas of Fukushima.

A First Generation Comparative Chromosome Map between Guinea Pig (Cavia porcellus) and Humans.

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.

Development of a sequential multicolor-FISH approach with 13 chromosome-specific painting probes for the rapid identification of river buffalo (Bubalus bubalis, 2n = 50) chromosomes

The development of new molecular techniques (array CGH, M-FISH, SKY-FISH, etc.) has led to great advancements in the entire field of molecular cytogenetics. However, the application of these methods is still very limited in farm animals. In the present study, we report, for the first time, the production of 13 river buffalo (Bubalus bubalis, 2n = 50) chromosome-specific painting probes, generated via chromosome microdissection and the DOP-PCR procedure. A sequential multicolor-FISH approach is also proposed on the same slide for the rapid identification of river buffalo chromosome/arms, namely, 1p-1q, 2p-2q, 3p-3q, 4p-4q, 5p-5q, 18, X, and Y, using both conventional and late-replicating banded chromosome preparations counterstained by DAPI. The provided 'bank' of chromosome-specific painting probes is useful for any further cytogenetic investigation not only for the buffalo breeds, but also for other species of the family Bovidae, such as cattle, sheep, and goats, for chromosome abnormality diagnosis, and, more generally, for evolutionary studies.

Molecular Cytogenetic Studies in Strepsirrhine Primates, Dermoptera and Scandentia

Since the first chromosome painting study between human and strepsirrhine primates was performed in 1996, nearly 30 species in Strepsirrhini, Dermoptera and Scandentia have been analyzed by cross-species chromosome painting. Here, the contribution of chromosome painting data to our understanding of primate genome organization, chromosome evolution and the karyotype phylogenetic relationships within strepsirrhine primates, Dermoptera and Scandentia is reviewed. Twenty-six to 43 homologous chromosome segments have been revealed in different species with human chromosome-specific paint probes. Various landmark rearrangements characteristic for each different lineage have been identified, as cytogenetic signatures that potentially unite certain lineages within strepsirrhine primates, Dermoptera and Scandentia.

Similar rates of chromosomal aberrant secondary oocytes in two indigenous cattle ( Bos taurus) breeds as determined by dual-color FISH

In vitro-matured metaphase II (MII) oocytes with corresponding first polar bodies (I pb) from two indigenous cattle ( Bos taurus) breeds have been investigated to provide specific data upon the incidence of aneuploidy. A total of 165 and 140 in vitro-matured MII oocytes of the Podolian (PO) and Maremmana (MA) breeds, respectively, were analyzed by fluorescence in situ hybridization using Xcen and five chromosome-specific painting probes. Oocytes with unreduced chromosome number were 13.3% and 6.4% in the two breeds, respectively, averaging 10.2%. In the PO, out of 100 MII oocytes + I pb analyzed, two oocytes were nullisomic for chromosome 5 (2.0%) and one disomic for the same chromosome (1.0%). In the MA, out of 100 MII oocytes + I pb, one oocyte was found nullisomic for chromosome 5 (1.0%) and one was disomic for the X chromosome (1.0%). Out of 200 MII oocytes + I pb, the mean rate of aneuploidy (nullisomy + disomy) for the two chromosomes scored was 2.5%, of which 1.5% was due to nullisomy and 1.0% due to disomy. By averaging these data with those previously reported on dairy cattle, the overall incidence of aneuploidy in cattle, as a species, was 2.25%, of which 1.25% was due to nullisomy and 1.0% due to disomy. The results so far achieved indicate similar rates of aneuploidy among the four cattle breeds investigated. Interspecific comparison between cattle (Xcen-5 probes) and pig ( Sus scrofa domestica) (1–10 probes) also reveal similar rates. Further studies are needed that use more probes to investigate the interchromosomal effect. Establishing a baseline level of aneuploidy for each species/breed could also be useful for improving the in vitro production of embryos destined to the embryo transfer industry as well as for monitoring future trends of the reproductive health of domestic animals in relation to management errors and/or environmental hazards.

Chromosomal evolution in Gekkonidae. I. Chromosome painting between Gekko and Hemidactylus species reveals phylogenetic relationships within the group

Geckos are a large group of lizards characterized by a rich variety of species, different modes of sex determination and diverse karyotypes. In spite of many unresolved questions on lizards' phylogeny and taxonomy, the karyotypes of most geckos have been studied by conventional cytogenetic methods only. We used flow-sorted chromosome-specific painting probes of Japanese gecko (Gekko japonicus), Mediterranean house gecko (Hemidactylus turcicus) and flat-tailed house gecko (Hemidactylus platyurus) to reveal homologous regions and to study karyotype evolution in seven gecko species (Gekko gecko, G. japonicus, G. ulikovskii, G. vittatus, Hemidactylus frenatus, H. platyurus and H. turcicus). Generally, the karyotypes of geckos were found to be conserved, but we revealed some characteristic rearrangements including both fissions and fusions in Hemidactylus. The karyotype of H. platyurus contained a heteromorphic pair in all female individuals, where one of the homologues had a terminal DAPI-negative and C-positive heterochromatic block that might indicate a putative sex chromosome. Among two male individuals studied, only one carried such a polymorphism, and the second one had none, suggesting a possible ZZ/ZW sex determination in some populations of this species. We found that all Gekko species have retained the putative ancestral karyotype, whilst the fission of the largest ancestral chromosome occurred in the ancestor of modern Hemidactylus species. Three common fissions occurred in the ancestor of Mediterranean house and flat-tailed house geckos, suggesting their sister group relationships. PCR-assisted mapping on flow-sorted chromosome libraries with conserved DMRT1 gene primers in G. japonicus indicates the localization of DMRT1 gene on chromosome 6.

Comparative Chromosome Maps of Neotropical Rodents Necromys lasiurus and Thaptomys nigrita (Cricetidae) Established by ZOO-FISH

This work presents chromosome homology maps between Mus musculus (MMU) and 2 South American rodent species from the Cricetidae group: Necromys lasiurus (NLA, 2n = 34) and Thaptomys nigrita (TNI, 2n = 52), established by ZOO-FISH using mouse chromosome-specific painting probes. Extending previous molecular cytogenetic studies in Neotropical rodents, the purpose of this work was to delineate evolutionary chromosomal rearrangements in Cricetidae rodents and to reconstruct the phylogenetic relationships among the Akodontini species. Our phylogenetic reconstruction by maximum parsimony analysis of chromosomal characters confirmed one consistent clade of all Neotropical rodents studied so far. In both species analyzed here, we observed the syntenic association of chromosome segments homologous to MMU 8/13, suggesting that this chromosome form is a synapomorphic trait exclusive to Neotropical rodents. Further, the previously described Akodontini-specific syntenic associations MMU 3/18 and MMU 6/12 were observed in N.lasiurus but not in T. nigrita, although the latter species is considered a member of the Akodontini tribe by some authors. Finally, and in agreement with this finding, N.lasiurus and Akodon serrensis share the derived fission of MMU 13, which places them as basal sister clades within Akodontini.

Karyotypic evolution in family Hipposideridae (Chiroptera, Mammalia) revealed by comparative chromosome painting, G- and C-banding.

Comparing to its sister-family (Rhinolophidae), Hipposideridae was less studied by cytogenetic approaches. Only a few high-resolution G-banded karyotypes have been reported so far, and most of the conclusions on the karyotypic evolution in Hipposideridae were based on conventional Giemsa-staining. In this study, we applied comparative chromosome painting, a method of choice for genome-wide comparison at the molecular level, and G- and C-banding to establish comparative map between five hipposiderid species from China, using a whole set of chromosome-specific painting probes from one of them (Aselliscus stoliczkanus). G-band and C-band comparisons between homologous segments defined by chromosome painting revealed that Robertsonian translocations, paracentric inversions and heterochromatin addition could be the main mechanism of chromosome evolution in Hipposideridae. Comparative analysis of the conserved chromosomal segments among five hipposiderid species and outgroup species suggests that bi-armed chromosomes should be included into the ancestral karyotype of Hipposideridae, which was previously believed to be exclusively composed of acrocentric chromosomes.

Reconstruction of the Putative Cervidae Ancestral Karyotype by Chromosome Painting of Siberian Roe Deer (Capreolus pygargus) with Dromedary Probes

The Siberian roe deer (Capreolus pygargus) is one of a few deer species presumably preserving the ancestral cervid karyotype. The comparative genomic data of the Siberian roe deer are critical for our understanding of the karyotypic relationships within artiodactyls. We have established chromosomal homologies between the Siberian roe deer and the dromedary (Camelus dromedarius) by cross-species chromosome painting with dromedary chromosome-specific painting probes. Dromedary chromosome paints detected 53 autosomal homologies in the genome of the Siberian roe deer. The identification of chromosomal homologies between the Siberian roe deer and cattle resulted from previously detected cattle-dromedary homologies. We have found 8 chromosomal rearrangements (6 fissions in the Siberian roe deer, 1 fission in the cattle and 1 inversion on the CPY11) that have separated the karyotypes of the cattle and the Siberian roe deer. The inversion on CPY11 might be an apomorphic trait of cervids, since we detected its presence in the gray brocket deer (Mazama gouazoubira). Thus our data further prove the scenario of chromosomal rearrangements that was previously proposed and add some new data.

Cross-Species Chromosome Painting Corroborates Microchromosome Fusion during Karyotype Evolution of Birds

The stone curlew, also known as thick-knee (Burhinus oedicnemus, BOE), represents a phylogenetically young species of the shorebirds (Charadriiformes) that exhibits one of the most atypical genome organizations known within the class of Aves, due to an extremely low diploid number (2n = 42) and only 6 pairs of microchromosomes in its complement. This distinct deviation from the ‘typical’ avian karyotype is attributed to repeated fusions of ancestral microchromosomes. In order to compare different species with this atypical avian karyotype and to investigate the chromosome rearrangement patterns, chromosome-specific painting probes representing the whole genome of the stone curlew were used to delineate chromosome homology between BOE and 5 species belonging to 5 different avian orders: herring gull (Charadriiformes), cockatiel (Psittaciformes), rock pigeon (Columbiformes), great gray owl (Strigiformes) and Eurasian coot (Gruiformes). Paints derived from the 20 BOE autosomes delimited 28 to 33 evolutionarily conserved segments in the karyotypes of the 5 species, similar to the number recognized by BOE paints in such a basal lineage as the chicken (28 conserved segments). This suggests a high degree of conservation in genome organization in birds. BOE paints also revealed some species-specific rearrangements. In particular, chromosomes BOE1–4 and 14, as well as to a large extent BOE5 and 6, showed conserved synteny with macrochromosomes, whereas homologous regions for BOE7–13 are found to be largely distributed on microchromosomes in the species investigated. Interestingly, the 6 pairs of BOE microchromosomes 15–20 appear to have undergone very few rearrangements in the 5 lineages investigated. Although the arrangements of BOE homologous segments on some chromosomes can be explained by complex fusions and inversions, the occurrence of homologous regions at multiple sites may point to fission of ancestral chromosomes in the karyotypes of the species investigated. However, the present results demonstrate that the ancestral microchromosomes most likely experienced fusion in the stone curlew lineage forming the medium-sized BOE chromosomes, while they have been conserved as microchromosomes in the other neoavian lineages.

Frequency of aneuploidy in in vitro–matured MII oocytes and corresponding first polar bodies in two dairy cattle ( Bos taurus) breeds as determined by dual-color fluorescent in situ hybridization

The current study was undertaken to investigate the aneuploidy rates in in vitro–matured meiosis II (MII) oocytes and corresponding first polar bodies in two dairy cattle ( Bos taurus) breeds by using dual-color fluorescent in situ hybridization (FISH). A total of 159 and 144 in vitro–matured MII oocytes of the Italian Friesian and Italian Brown breeds, respectively, were obtained according to the standard methods and analyzed by FISH using “Xcen” and “5” chromosome-specific painting probes, produced by chromosome microdissection and Degenerate Oligonucleotide Primer- Polymerase Chain Reaction (DOP-PCR). Oocytes with unreduced chromosome number were 10.1% and 16.7% in the two breeds, respectively. To avoid bias due to possible artifacts, the aneuploidy rates were determined by analyzing only oocytes with the corresponding polar bodies. In the Italian Friesian, 100 of 143 (69.9%) secondary MII oocytes showed clear MII plates with corresponding first polar bodies and were scored for aneuploidy detection; one oocyte was “nullisomic” for chromosome X (1.0%) and one “disomic” for chromosome 5 (1.0%). In the Italian Brown, 100 of 120 (83.3%) MII oocytes with corresponding first polar bodies were analyzed; one oocyte was nullisomic (1.0%) and one was disomic (1.0%), both for chromosome 5. Totally, 303 oocytes were analyzed, 40 of which showed an unreduced chromosome complement (13.2%); of 200 MII oocytes with the corresponding first polar bodies, the aneuploidy rate (nullisomy + disomy) for the two chromosomes scored was 2%. Assuming that each chromosome is equally involved in aneuploidy, it results that in cattle oocytes matured in vitro, at least 30% of the oocytes (1 × 30 haploid chromosomes) should be aneuploid. Premature separation of sister chromatids (PSSC) was also observed in 2% of the oocytes in the Italian Friesian breed involving chromosome 5 and in 1% of the Italian Brown breed involving the X chromosome. Estimation of the “baseline” level of aneuploidy in the in vitro–matured oocytes of the various domestic animal species and breeds is, to our opinion, a useful reference for improving the in vitro production of embryos as well as for monitoring future trends of the reproductive health of the species/breeds engaged in zootechnical productions, especially in relation to management errors and environmental hazards.

X-Y Sperm Aneuploidy in 2 Cattle (Bos taurus) Breeds as Determined by Dual Color Fluorescent in situ Hybridization (FISH)

The present study was undertaken to investigate aneuploidy rates in the sperm populations of 2 cattle (Bos taurus) breeds by using dual color fluorescent in situ hybridization (FISH) with Xcen and Y chromosome-specific painting probes, obtained by chromosome microdissection and DOP-PCR. Frozen semen from 10 Italian Friesian and 10 Italian Brown testing bulls was used for the investigation. For each bull, more than 5,000 sperm were analyzed, for a total of 52,586 and 51,342 sperm cells for the 2 breeds, respectively. The present study revealed – in both breeds – a preponderance of the Y-bearing sperm compared to the X-bearing sperm. Within each breed, a statistically significant variation in the various classes of aneuploidy (XX, YY and XY) was found: differences were found in the Friesian breed among the 3 diploidy classes, and in the Brown breed, among the 3 disomy classes (p < 0.05) as well as among the 3 diploidy classes (p < 0.01). However, the 2 breeds did not differ significantly in the overall mean rates of X-Y aneuploidy (disomy + diploidy) which amounts to 0.162% in the Italian Friesian and 0.142% in the Italian Brown. When meiosis I (MI) and II (MII) errors were compared, statistically significant differences (p < 0.01) were found in the disomy classes and in both breeds, whereas the differences between diploidy classes were not significant. Compared to humans, a lower level of aneuploidy has been found in the domestic species analyzed so far. The present study contributes to the establishment of a baseline level of aneuploidy in the sperm populations of 2 cattle breeds which could be used for monitoring future trends of reproductive health, especially in relation to environmental changes and mutagens.