Intact rDNA arrays of Potentilla-origin detected in Erythronium nucleus suggest recent eudicot-to-monocot horizontal transfer

The occurrence of horizontal gene transfer (HGT) in Eukarya is increasingly gaining recognition. Nuclear-to-nuclear jump of DNA between plant species at high phylogenetic distance and devoid of intimate association (e.g., parasitism) is still scarcely reported. Within eukaryotes, components of ribosomal DNA (rDNA) multigene family have been found to be horizontally transferred in protists, fungi and grasses. However, in neither case HGT occurred between phylogenetic families, nor the transferred rDNA remained tandemly arrayed and transcriptionally active in the recipient organism. This study aimed to characterize an alien eudicot-type of 45S nuclear rDNA, assumingly transferred horizontally to the genome of monocot European Erythronium (Liliaceae). Genome skimming coupled by PacBio HiFi sequencing of a BAC clone were applied to determine DNA sequence of the alien rDNA. A clear phylogenetic signal traced the origin of the alien rDNA of Erythronium back to the Argentea clade of Potentilla (Rosaceae) and deemed the transfer to have occurred in the common ancestor of E. dens-canis and E. caucasicum. Though being discontinuous, transferred rDNA preserved its general tandemly arrayed feature in the host organism. Southern blotting, molecular cytogenetics, and sequencing of a BAC clone derived from flow-sorted nuclei indicated integration of the alien rDNA into the recipient's nuclear genome. Unprecedently, dicot-type alien rDNA was found to be transcribed in the monocot Erythronium albeit much less efficiently than the native counterpart. This study adds a new example to the growing list of naturally transgenic plants while holding the scientific community continually in suspense about the mode of DNA transfer.

Further insights on chromosomal evolution of the genus Enyalius with karyotype description of Enyalius boulengeri Etheridge, 1969 (Squamata, Leiosauridae)

The genus Enyalius is composed of 10 described species inhabiting forest areas in Amozônia, Cerrado and Atlantic forest biomes. Currently, eight species with high levels of chromosome variation have been karyotyped. The study aims to characterize the karyotype of Enyalius boulengeri, with classical and molecular techniques, and improve knowledge about the karyotype evolution of the lizard genus Enyalius. The species has 2n = 36 chromosomes (8m + 4sm + 24mc), FN = 24; NORs and 18S rDNA were subtelomeric and located on chromosome pair 2. Repetitive DNA probes (CAT)10 accumulated on centromeric and terminal regions of some macrochromosomes. (GA)15 probe showed conspicuous accumulation on the pericentromeric region of chromosome pairs 1 and 6. Repetitive FISH patterns obtained with (GC)15 probe marked the pericentromeric region of the first chromosome pair. All probes showed accumulation in the microchromosomes. The chromosomal formula found on E. boulengeri has been considered the ancestral karyotype for pleurodont Iguania. The genus Enyalius is characterized by two distinctive chromosomal groups; one with highly conserved karyotypes, whereas the other is karyotypically diverse. Our molecular cytogenetics data are promising and will increase knowledge about the genus Enyalius chromosome evolution.

Chromosome and Genome Diversity in the Genus Trifolium (Fabaceae)

Trifolium L. is an economically important genus that is characterized by variable karyotypes relating to its ploidy level and basic chromosome numbers. The advent of genomic resources combined with molecular cytogenetics provides an opportunity to develop our understanding of plant genomes in general. Here, we summarize the current state of knowledge on Trifolium genomes and chromosomes and review methodologies using molecular markers that have contributed to Trifolium research. We discuss possible future applications of cytogenetic methods in research on the Trifolium genome and chromosomes.

Molecular Cytogenetics in the Era of Chromosomics and Cytogenomic Approaches

Here the role of molecular cytogenetics in the context of yet available all other cytogenomic approaches is discussed. A short introduction how cytogenetics and molecular cytogenetics were established is followed by technical aspects of fluorescence in situ hybridization (FISH). The latter contains the methodology itself, the types of probe- and target-DNA, as well as probe sets. The main part deals with examples of modern FISH-applications, highlighting unique possibilities of the approach, like the possibility to study individual cells and even individual chromosomes. Different variants of FISH can be used to retrieve information on genomes from (almost) base pair to whole genomic level, as besides only second and third generation sequencing approaches can do. Here especially highlighted variations of FISH are molecular combing, chromosome orientation-FISH (CO-FISH), telomere-FISH, parental origin determination FISH (POD-FISH), FISH to resolve the nuclear architecture, multicolor-FISH (mFISH) approaches, among other applied in chromoanagenesis studies, Comet-FISH, and CRISPR-mediated FISH-applications. Overall, molecular cytogenetics is far from being outdated and actively involved in up-to-date diagnostics and research.

Molecular cytogenetics of Hedysarum gmelinii Ledeb. and H. setigerum Turcz. (Fabaceae) from populations of Southern Siberia

For the first time, a comparative karyotype analysis of closely related species Hedysarum gmelinii andH. setigerum (Hedysarum section Multicaulia) grown in Southern Siberia, has been performed by molecular cytogeneticmarkers. Chromosome numbers in karyotypes of these species were specified – 2n = 4х = 32. In some accessions, additionalB chromosomes were revealed. FISH analyses indicated high similarities in chromosome morphology and also patternsof chromosomal distributions of 45S and 5S rDNA clusters in karyotypes of H. gmelinii and H. setigerum, which confirmsthe close relationship between their genomes.

Karyotype structure and cytogenetic markers of Amoimyrmex bruchi and Amoimyrmex silvestrii: the contribution to understanding leafcutting ant relationships

Leafcutting ants are considered the most important herbivores in terrestrial environments throughout the Neotropics. <i>Amoimyrmex</i> is the sister clade of the remaining leafcutter ants from the genera <i>Atta</i> and <i>Acromyrmex</i>. <i>Amoimyrmex striatus</i> was the only species cytogenetically studied within the genus and shares the same chromosomal number with <i>Atta</i>, bearing 22 chromosomes, whereas <i>Acromyrmex</i> bears 38 chromosomes, with the exception of the social parasite <i>Acromyrmex ameliae</i> (2n=36). Our objective here was to analyze cytogenetically the species of <i>Amoimyrmex bruchi</i> and <i>Amoimyrmex silvestrii</i>, as well as to describe the karyotype of these sister species, by means of an integrative approach using classical and molecular cytogenetics. We aimed to characterize cytogenetic markers that contribute to the systematics and taxonomy of the genus. Our results showed that the karyotypes of these two species are very similar, with an identical chromosome number (2n=22), chromosome morphology (2K=20m+2sm), and location of 18S rDNA and the telomeric repeat TTAGG on the chromosomes. Yet, the microsatellite probe GA(15) showed variation across the species and populations studied. We suggest that both species diverged relatively recently and are unmistakably sisters because of the many shared characteristics, including the highly conserved karyotypes.

Prenatal Diagnosis of True Fetal Mosaicism with Small Supernumerary Marker Chromosome Derived from Chromosome 16 by Funipuncture and Molecular Cytogenetics Including Chromosome Microarray

This study examined the molecular characterization of a prenatal case with true fetal mosaicism of small supernumerary marker chromosome 16 (sSMC(16)). A 41-year-old female underwent amniocentesis at 19 weeks of gestation due to advanced maternal age. Chromosomal analysis for cultured amniocytes revealed a karyotype of 47,XY,+mar[4]/46,XY[16]. Spectral karyotyping and metaphase fluorescence in situ hybridization (FISH) demonstrated that the sSMC was derived from chromosome 16 (47,XY,+mar.ish der(16)(D16Z1+)[13/20]). Confined placental mosaicism was initially suspected because the prenatal ultrasound revealed a normal structure and the pregnancy was uneventful. However, interphase FISH of cord blood performed at 28 weeks of gestation showed 20% mosaicism of trisomy chromosome 16 (nuc ish(D16Z2×3)[40/200]). Chromosome microarray analysis further demonstrated 55% mosaicism of an 8.02 Mb segmental duplication at the subcentromeric region of 16p12.1p11.1 (arr[GRCh37] 16p12.1p11.1(27021975_35045499)×3[0.55]). The results demonstrated a true fetal mosaicism of sSMC(16) involving chromosome16p12.1p11.1 that is associated with chromosome 16p11.2 duplication syndrome (OMIM #614671). After non-directive genetic counseling, the couple opted for late termination of pregnancy. This case illustrated the use of multiple molecular cytogenetic tools to elucidate the origin and structure of sSMC, which is crucial for prenatal counseling, decision making, and clinical management.

Karyotype asymmetry in Cuscuta L. subgenus Pachystigma reflects its repeat DNA composition

Cuscuta is a cytogenetically diverse genus, with karyotypes varying 18-fold in chromosome number and 89-fold in genome size. Each of its four subgenera also presents particular chromosomal features, such as bimodal karyotypes in Pachystigma. We used low coverage sequencing of the Cuscuta nitida genome (subgenus Pachystigma), as well as chromosome banding and molecular cytogenetics of three subgenus representatives, to understand the origin of bimodal karyotypes. All three species, C. nitida, C. africana (2n = 28) and C. angulata (2n = 30), showed heterochromatic bands mainly in the largest chromosome pairs. Eighteen satellite DNAs were identified in C. nitida genome, two showing similarity to mobile elements. The most abundant were present at the largest pairs, as well as the highly abundant ribosomal DNAs. The most abundant Ty1/Copia and Ty3/Gypsy elements were also highly enriched in the largest pairs, except for the Ty3/Gypsy CRM, which also labelled the pericentromeric regions of the smallest chromosomes. This accumulation of repetitive DNA in the larger pairs indicates that these sequences are largely responsible for the formation of bimodal karyotypes in the subgenus Pachystigma. The repetitive DNA fraction is directly linked to karyotype evolution in Cuscuta.

Application of the FISH Technique to Visualize Sex Chromosomes in Domestic Cat Spermatozoa

Fluorescence in situ hybridization is a molecular cytogenetics technique that enables the visualization of chromosomes in cells via fluorescently labeled molecular probes specific to selected chromosomes. Despite difficulties in carrying out the FISH technique on sperm, related to the need for proper nuclear chromatin decondensation, this technique has already been used to visualize chromosomes in human, mouse, cattle, swine, horse, and dog spermatozoa. Until now, FISH has not been performed on domestic cat sperm; therefore, the aim of this study was to visualize sex chromosomes in domestic cat sperm. The results showed the presence of X and Y chromosomes in feline spermatozoa. The procedure used for sperm decondensation and fluorescence in situ hybridization was adequate to visualize chromosomes in domestic cat spermatozoa and, in the future, it may be used to determine the degree of chromosomal abnormalities in these gametes.