Cytogenetic analyses of Arabidopsis.

For a long time, Arabidopsis thaliana was not a favorite of cytogeneticists because of its small chromosomes. Nevertheless, most cytogenetic approaches have become applicable to this model plant during the past decade. Recently, the very small genome, the low content, and strong clustering of repetitive sequences even turned out to be advantageous for the first establishment of chromosome painting in a euploid plant. Chromosome painting together with the high-resolution fluorescence in situ hybridization (FISH) mapping on Arabidopsis pachytene chromosomes has opened new perspectives for plant cytogenetics. In this chapter, we describe standard cytogenetic methods including preparation of chromosomes and chromatin fibers, isolation of interphase nuclei, FISH and immunostaining, painting of Arabidopsis chromosomes, and comparative chromosome painting in other Brassicaceae species.

Lysak M ，Fransz P ，Schubert I 《-》

Chromosome landmarks as tools to study the genome of Arabidopsis thaliana.

The model plant Arabidopsis thaliana has long been used for genetic, cellular and molecular studies. Whereas this plant was used as a model of genetics in the 1940's, the first cytogenetic observation of A. thaliana chromosomes was published in the beginning of the 20th century. Although Arabidopsis was not originally considered to be a good plant model for cytogenetics due to smallness of its genome, the number of published chromosome studies has expanded enormously in recent years. The advent of fluorescence in situ hybridization techniques on meiotic chromosomes together with indirect immuno-fluorescence localization of key chromosomal and nuclear proteins and wide accessibility of Arabidopsis mutants have resulted in a synergistic boost in Arabidopsis cytogenetics. In comparison to other plant species, the small genome with under-represented DNA repeats together with a small number of chromosomes makes this model plant easy to comprehend for a cytologist.

Siroky J 《-》

Reciprocal and multi-species chromosome BAC painting in crucifers (Brassicaceae).

In crucifer cytogenomics, BAC contigs of Arabidopsis thaliana have been used as probes for comparative chromosome painting among species. Here we successfully tested chromosome-specific BAC contigs of A. thaliana (n = 5) and A. halleri (n = 8) as probes for reciprocal BAC painting. Furthermore, BAC contigs of both Arabidopsis species were applied as multi-species painting probes to a third crucifer species, Noccaea caerulescens (n = 7), revealing their shared chromosome homeology. Specifically, we found homeology across portions of chromosomes At2, Ah4, and Nc4, which reflects their shared common origin with chromosome AK4 of the Ancestral Crucifer Karyotype (n = 8). We argue that multi-species and multi-directional painting will significantly expedite comparative cytogenomics in Brassicaceae and other plant families.

Lysak MA ，Mandáková T ，Lacombe E 《-》

Fluorescence in situ hybridization for molecular cytogenetic analysis in filamentous fungi.

Fluorescence in situ hybridization (FISH) is a powerful technology for studying eukaryotic chromosomes and genomes from the combined view of cytogenetics and molecular biology, but its use in filamentous fungi has been limited. In this chapter, we describe protocols to perform three basic FISH techniques in filamentous fungi: (a) FISH mapping of unique sequences on the somatic chromosomes and interphase nuclei, (b) chromosome painting to detect a specific chromosome in the genome by fluorescent painting of the whole chromosome, and (c) fiber FISH on the stretched DNA fibers for physical mapping. The ways of preparing target specimens unique to filamentous fungi are included in the protocols.

Tsuchiya D ，Taga M 《-》

Methods for Cytogenetic Chromosome Barcoding and Chromosome Painting in Brachypodium distachyon and Its Relative Species.

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.

Idziak-Helmcke D ，Betekhtin A 《-》