Abstract: The chromosome arrangement in interphase nuclei is of growing interest, e.g., the spatial vicinity of homologous sequences is decisive for efficient repair of DNA damage by homologous recombination, and close alignment of sister chromatids is considered as a prerequisite for their bipolar orientation and subsequent segregation during nuclear division. To study the degree of homologous pairing and of sister chromatid alignment in plants, we applied fluorescent in situ hybridisation with specific bacterial artificial chromosome inserts to interphase nuclei. Previously we found in Arabidopsis thaliana and in A. lyrata positional homologous pairing at random, and, except for centromere regions, sister chromatids were frequently not aligned. To test whether these features are typical for higher plants or depend on genome size, chromosome organisation and/or phylogenetic affiliation, we investigated distinct individual loci in other species. The positional pairing of these loci was mainly random. The highest frequency of sister alignment (in >93% of homologues) was found for centromeres, some rDNA and a few other high copy loci. Apparently, somatic homologous pairing is not a typical feature of angiosperms, and sister chromatid aligment is not obligatory along chromosome arms. Thus, the high frequency of chromatid exchanges at homologous positions after mutagen treatment needs another explanation than regular somatic pairing of homologues (possibly an active search of damaged sites for homology). For sister chromatid exchanges a continuous sister chromatid alignment is not required. For correct segregation, permanent alignment of sister centromeres is sufficient.

Abstract: Whole-mount fluorescence in situ hybridization (FISH) was applied to Arabidopsis thaliana seedlings to determine the three-dimensional (3D) interphase chromosome territory (CT) arrangement and heterochromatin location within the positional context of entire tissues or in particular cell types of morphologically well-preserved seedlings. The interphase chromosome arrangement was found to be similar between all inspected meristematic and differentiated root and shoot cells, indicating a lack of a gross reorganization during differentiation. The predominantly random CT arrangement (except for a more frequent association of the homologous chromosomes bearing a nucleolus organizer) and the peripheric location of centromeric heterochromatin were as previously observed for flow-sorted nuclei, but centromeres tend to fuse more often in nonendoreduplicating cells and NORs in differentiated cells. After mitosis, sister nuclei revealed a symmetric arrangement of homologous CTs waning with the progress of the cell cycle or in the course of differentiation. Thus, the interphase chromosome arrangement in A. thaliana nuclei seems to be constrained mainly by morphological features such as nuclear shape, presence or absence of a nucleolus organizer on chromosomes, nucleolar volume, and/or endopolyploidy level.

Abstract: Evolution of chromosome complements can be resolved by genome sequencing, comparative genetic mapping, and comparative chromosome painting. Previously, comparison of genetic maps and gene-based phylogenies suggested that the karyotypes of Arabidopsis thaliana (n = 5) and of related species with six or seven chromosome pairs were derived from an ancestral karyotype with eight chromosome pairs. To test this hypothesis, we applied multicolor chromosome painting using contiguous bacterial artificial chromosome pools of A. thaliana arranged according to the genetic maps of Arabidopsis lyrata and Capsella rubella (both n = 8) to A. thaliana, A. lyrata, Neslia paniculata, Turritis glabra, and Hornungia alpina. This approach allowed us to map the A. lyrata centromeres as a prerequisite to defining a putative ancestral karyotype (n = 8) and to elucidate the evolutionary mechanisms that shaped the karyotype of A. thaliana and its relatives. We conclude that chromosome "fusions" in A. thaliana resulted from (i) generation of acrocentric chromosomes by pericentric inversions, (ii) reciprocal translocation between two chromosomes (one or both acrocentric), and (iii) elimination of a minichromosome that arose in addition to the "fusion chromosome." Comparative chromosome painting applied to N. paniculata (n = 7), T. glabra (n = 6), and H. alpina (n = 6), for which genetic maps are not available, revealed chromosomal colinearity between all species tested and allowed us to reconstruct the evolution of their chromosomes from a putative ancestral karyotype (n = 8). Although involving different ancestral chromosomes, chromosome number reduction followed similar routes as found within the genus Arabidopsis.

Abstract: In contrast to the situation described for mammals and Drosophila, chromosome territory (CT) arrangement and somatic homologous pairing in interphase nuclei of Arabidopsis thaliana (n = 5) are predominantly random except for a more frequent association of the chromosomes bearing a homologous nucleolus organizer region. To find out whether this chromosome arrangement is also characteristic for other species of the genus Arabidopsis, we investigated Arabidopsis lyrata ssp. lyrata (n = 8), one of the closest relatives of A. thaliana. First, we determined the size of each chromosome and chromosome arm, the sequence type of centromeric repeats and their distribution between individual centromeres and the position of the 5S/45S rDNA arrays in A. lyrata. Then we demonstrated that CT arrangement, homologous pairing and sister chromatid alignment of distinct euchromatic and/or heterochromatic regions within A. lyrata interphase nuclei are similar to that in A. thaliana nuclei. Thus, the arrangement of interphase chromosomes appears to be conserved between both taxa that diverged about 5 million years ago. Since the chromosomes of A. lyrata resemble those of the presumed ancestral karyotype, a similar arrangement of interphase chromosomes is also to be expected for other closely related diploid species of the Brassicaceae family.

Abstract: Efficient amplification and labelling of probes are crucial for successful sequence detection by fluorescent in situ hybridization (FISH). In particular, chromosome painting to visualize chromosome segments or entire chromosomes by FISH requires large amounts of probes derived from extended templates. There are a number of techniques for probe labelling. The most widespread is nick translation, based on the replicational incorporation of modified nucleotides. Here we demonstrate successful rolling-circle amplification (RCA) of very low amounts of long circular template sequences (single bacterial artificial chromosomes (BACs) or pools of BACs). The amplicons were suitable for labelling by nick translation and subsequent FISH. A novel achievement is the use of RCA for simultaneous amplification and labelling of single BACs or BAC pools in a labour- and cost-effective manner.