Halina Cernohorska

The use of laser microdissection for the preparation of chromosome-specific painting probes in farm animals.

Laser microbeam microdissection and laser pressure catapulting procedure were used for the construction of chromosome-specific painting probes, arm-specific probes and probes for chromosomal subfragments. We report on a method for generation of fluorescence in-situ hybridization probes from laser dissected chromosomes of farm animals. So far, using the described method, a set of chromosome-specific painting probes has been obtained for all porcine chromosomes, 17 chromosomes of cattle and selected equine chromosomes. It is concluded that the laser technology appears to be a useful and powerful tool for the construction of chromosome-specific painting probes. Its main advantage is the fast non-contact collection of chromosomes.

FAST-FISH with laser beam microdissected DOP-PCR probe distinguishes the sex chromosomes of Silene latifolia

We present an improved FISH strategy for differentiating the sex chromosomes of the dioecious model plant, Silene latifolia. Fixed mitotic protoplasts were dropped on a polyethylene naphthalate membrane, the X or Y chromosomes were isolated using nitrogen laser beam microdissection, catapulted by laser pressure, and amplified by DOP-PCR. A modified FAST-FISH protocol based on a short hybridization time combined with a low concentration of probe was used. The success of this approach is demonstrated by the differential labeling of the X and Y chromosomes and it could represent a quick method for comparing organization of plant genomes.

Phylogenomic study of spiral-horned antelope by cross-species chromosome painting

Chromosomal homologies have been established between cattle (Bos taurus, 2nu2009=u200960) and eight species of spiral-horned antelope, Tribe Tragelaphini: Nyala (Tragelaphus angasii, 2nu2009=u200955♂/56♀), Lesser kudu (T. imberbis, 2nu2009=u200938♂,♀), Bongo (T. eurycerus, 2nu2009=u200933♂/34♀), Bushbuck (T. scriptus, 2nu2009=u200933♂/34♀), Greater kudu (T. strepsiceros, 2nu2009=u200931♂/32♀), Sitatunga (T. spekei, 2nu2009=u200930♂,♀) Derby eland (Taurotragus derbianus 2nu2009=u200931♂/32♀) and Common eland (T. oryx 2nu2009=u200931♂/32♀). Chromosomes involved in centric fusions in these species were identified using a complete set of cattle painting probes generated by laser microdissection. Our data support the monophyly of Tragelaphini and a clade comprising T. scriptus, T. spekei, T. euryceros and the eland species T. oryx and T. derbianus, findings that are largely in agreement with sequence-based molecular phylogenies. In contrast, our study suggests that the arid adaptiveness of T. oryx and T. derbianus is recent. Finally, we have identified the presence of the rob(1;29) fusion as an evolutionary marker in most of the tragelaphid species investigated. This rearrangement is associated with reproductive impairment in cattle and raises questions whether subtle distinctions in breakpoint location or differential rescue during meiosis underpin the different outcomes detected among these lineages.

Efficient high-throughput sequencing of a laser microdissected chromosome arm.

BackgroundGenomic sequence assemblies are key tools for a broad range of gene function and evolutionary studies. The diploid amphibian Xenopus tropicalis plays a pivotal role in these fields due to its combination of experimental flexibility, diploid genome, and early-branching tetrapod taxonomic position, having diverged from the amniote lineage ~360 million years ago. A genome assembly and a genetic linkage map have recently been made available. Unfortunately, large gaps in the linkage map attenuate long-range integrity of the genome assembly.ResultsWe laser dissected the short arm of X. tropicalis chromosome 7 for next generation sequencing and computational mapping to the reference genome. This arm is of particular interest as it encodes the sex determination locus, but its genetic map contains large gaps which undermine available genome assemblies. Whole genome amplification of 15 laser-microdissected 7p arms followed by next generation sequencing yielded ~35 million reads, over four million of which uniquely mapped to the X. tropicalis genome. Our analysis placed more than 200 previously unmapped scaffolds on the analyzed chromosome arm, providing valuable low-resolution physical map information for de novo genome assembly.ConclusionWe present a new approach for improving and validating genetic maps and sequence assemblies. Whole genome amplification of 15 microdissected chromosome arms provided sufficient high-quality material for localizing previously unmapped scaffolds and genes as well as recognizing mislocalized scaffolds.

Comparative Molecular Cytogenetics in Cetartiodactyla

Cetartiodactyla comprises Artiodactyla (even-toed ungulates) and Cetacea (whales, dolphins and porpoises). Artiodactyla is a large taxon represented by about 200 living species ranked in 10 families. Cetacea are classified into 13 families with almost 80 species. Many publications concerning karyotypic relationships in Cetartiodactyla have been published in previous decades. Formerly, the karyotypes of closely related species were compared by chromosome banding. Introduction of molecular cytogenetic methods facilitated comparative mapping between species with highly rearranged karyotypes and distantly related species. Such information is a prerequisite for the understanding of karyotypic phylogeny and the reconstruction of the karyotypes of common ancestors. This study summarizes the data on chromosome evolution in Cetartiodactyla, mainly derived from molecular cytogenetic studies. Traditionally, phylogenetic relationships of most groups have been estimated using morphological data. However, the results of some molecular studies of mammalian phylogeny are discordant with traditional conceptions of phylogeny. Cetartiodactyls provide several examples of incongruence between traditional morphological and molecular data. Such cases of conflict include the relationships of the major clades of artiodactyls, the relationships among the extant families of the suborder Ruminantia or the phylogeny of the family Bovidae. The most unexpected aspect of the molecular phylogeny was the recognition that Cetacea is a deeply nested member of Artiodactyla. The largest living order of terrestrial hoofed mammals is the even-toed hoofed mammals, or Artiodactyla. The artiodactyls are composed of over 190 living species including pigs, peccaries, hippos, camels, llamas, deer, pronghorns, giraffes, sheep, goats, cattle and antelopes. Cetacea is an order of wholly aquatic mammals, which include whales, dolphins and porpoises. Cetartiodactyla has become the generally accepted name for the clade containing both of these orders.

A paradox revealed: karyotype evolution in the four-horned antelope occurs by tandem fusion (Mammalia, Bovidae, Tetracerus quadricornis).

The four-horned antelope, Tetracerus quadricornis, is a karyotypic novelty in Bovidae since chromosomal evolution in this species is driven by tandem fusions in contradiction to the overwhelming influence of Robertsonian fusions in other species within the family. Using a combination of differential staining and molecular cytogenetic techniques, we provide the first description of the species’ karyotype, draw phylogenetic inferences from the cytogenetic data and discuss possible mechanisms underlying the formation of the tandem fusions in this species. We show (a) that pairs 1–6 of Tetracerus correspond to a combination of Bos taurus orthologous chromosomes that are tandemly fused head to tail, (b) the presence of interstitial centromeric satellite DNA at the junctions of orthologous blocks defined by the cross-species painting data and (c) that in some instances, residual telomeric sequences persist at these sites. We conclude that the attendant result of each fusion is an enlarged acrocentric fusion element comprising a single functional centromere and two terminal telomeres that, collectively, led to a reduction of the 2nu2009=u200958 bovid ancestral acrocentric chromosomal complement to the 2nu2009=u200938 detected in the four-horned antelope.

Karyotype, centric fusion polymorphism and chromosomal aberrations in captive-born mountain reedbuck (Redunca fulvorufula)

Chromosomes of fourteen captive-born mountain reedbucks (Redunca fulvorufula) have been investigated. The diploid chromosome number was 2n = 56 (FN = 60). The mountain reedbuck karyotype consists of 26 acrocentric and two biarmed chromosome pairs resulting from two centric fusions involving chromosomes 2 and 25, and 6 and 10, respectively. In some animals, 57 chromosomes were detected. Variation in the diploid number was found to be due to polymorphism for the centric fusion 6;10. Both X and Y chromosomes are large and acrocentric. The entire Y chromosome and the proximal part of the X chromosome consist of heterochromatin. The chromosomes X, 9 and 14 appeared to be of caprine type. Chromosome aberrations have been detected in two of the 14 animals investigated. A de novo formed Robertsonian translocation rob(6;13) was found in one female heterozygous for the fusion 6;10. CBG-banding revealed one block of centromeric heterochromatin in the de novo formed translocation rob(6;13) and also in the evolutionarily fixed centric fusions 6;10 and 2;25. One examined male homozygous for fusion 6;10, had a mosaic 56,XY/57,XYY karyotype, with 11% of analyzed cells containing two Y chromosomes. The findings were confirmed by cross-species fluorescence in situ hybridization (FISH) with bovine (Bos taurus L.) chromosome painting probes. The study demonstrates the relevance of cytogenetic screening in captive animals from zoological gardens.

A Large Pseudoautosomal Region on the Sex Chromosomes of the Frog Silurana tropicalis

Sex chromosome divergence has been documented across phylogenetically diverse species, with amphibians typically having cytologically nondiverged (“homomorphic”) sex chromosomes. With an aim of further characterizing sex chromosome divergence of an amphibian, we used “RAD-tags” and Sanger sequencing to examine sex specificity and heterozygosity in the Western clawed frog Silurana tropicalis (also known as Xenopus tropicalis). Our findings based on approximately 20 million genotype calls and approximately 200 polymerase chain reaction-amplified regions across multiple male and female genomes failed to identify a substantially sized genomic region with genotypic hallmarks of sex chromosome divergence, including in regions known to be tightly linked to the sex-determining region. We also found that expression and molecular evolution of genes linked to the sex-determining region did not differ substantially from genes in other parts of the genome. This suggests that the pseudoautosomal region, where recombination occurs, comprises a large portion of the sex chromosomes of S. tropicalis. These results may in part explain why African clawed frogs have such a high incidence of polyploidization, shed light on why amphibians have a high rate of sex chromosome turnover, and raise questions about why homomorphic sex chromosomes are so prevalent in amphibians.

A comparative study of meiotic recombination in cattle (Bos taurus) and three wildebeest species (Connochaetes gnou, C. taurinus taurinus and C. t. albojubatus).

The karyotypic evolution in the family Bovidae is based on centric fusions of ancestral acrocentric chromosomes. Here, the frequency and distribution of meiotic recombination was analyzed in pachytene spermatocytes from Bos taurus (2n = 60) and 3 wildebeest species (Connochaetes gnou, C. taurinus taurinus and C. t. albojubatus) (2n = 58) using immunofluorescence and fluorescence in situ hybridization. Significant differences in mean numbers of recombination events per cell were observed between B. taurus and members of the genus Connochaetes (47.2 vs. 43.7, p < 0.001). The number of MLH1 foci was significantly correlated with the length of the autosomal synaptonemal complexes. The average interfocus distance was influenced by interference. The male recombination maps of bovine chromosomes 2 and 25 and of their fused homologues in wildebeests were constructed. A significant reduction of recombination in the fused chromosome BTA25 was observed in wildebeests (p = 0.005). This was probably caused by interference acting across the centromere, which was significantly stronger than the intra-arm interference. This comparative meiotic study showed significant differences among the species from the family Bovidae with the same fundamental number of autosomal arms (FNa = 29) which differ by a single centric fusion.

Molecular Insights into X;BTA5 Chromosome Rearrangements in the Tribe Antilopini (Bovidae)

For a clade that includes Antilope, Gazella,Nanger and Eudorcas (Antilopinae), X;BTA5 translocation is a synapomorphy. Using a combination of fluorescence in situ hybridization (FISH) probes and polymerase chain reaction techniques, we provide (i) the first insight into the X;BTA5 architecture which differs in the species under study: Antilope cervicapra (genus Antilope), Gazella leptoceros (genus Gazella) and Nanger dama ruficollis (genus Nanger), (ii) determination of interstitial satellite DNA at the X;BTA5 junctions, and (iii) determination of repetitive sequences occupying constitutive heterochromatin of Xp arms in the studied species. The distribution of 2 repetitive DNA families in the centromeric regions of all chromosomes has been investigated by FISH with probes representing satellite I and satellite II DNA in all studied species. In this context, we discuss a markedly smaller centromere in the BTA5 (Y2) unfused chromosomes in males in the XY1Y2 determining system in comparison with other acrocentrics. An analysis of karyotypic data described in current published studies revealed a disparity with the data determined by FISH. In this report, we document chromosomal fusions in the 3 species mentioned resulting from FISH with painting probes prepared from cattle (Bos taurus). The number and chromosomal location of nucleolus organizer regions were determined by FISH. In the present study, we emphasize the importance of chromosomal rearrangement verification, particularly, if they are used for phylogenetic analysis.