Comet-FISH for the evaluation of plant DNA damage after mutagenic treatments

The aim of this study was to perform a comparative investigation of the actions of three mutagens that are widely used in plant mutagenesis using the comet-FISH technique. The comet-FISH technique was used for the analysis of DNA damage and the kinetics of repair within specific DNA sequences. FISH with rDNA and telomeric/centromeric DNA probes was applied to comets that were obtained from an alkaline/neutral comet assay. Migration within specific DNA sequences was analysed after treatment with two chemical mutagens-maleic hydrazide (MH) and N-nitroso-N-methylurea (MNU), and γ-rays. Barley was used as a model plant in this study. The possible utility of specific DNA sequences in a comparative assessment of the distribution of DNA damage within a plant genome was evaluated. This study proved that the comet-FISH technique is suitable for a detailed quantification of DNA damage and repair within specific DNA sequences in plant mutagenesis. The analysis of FISH signals demonstrated that the involvement of specific DNA sequences in DNA damage was different and was dependent on the mutagen used. We showed that 5S rDNA and telomeric DNA sequences are more sensitive to mutagenic treatment, which was expressed by a stronger fragmentation and migration in comparison to the other probes used in the study. We found that 5S rDNA and telomeric DNA probes are more suitable for testing the genotoxicity of environmental factors. A comparison of the involvement of specific chromosome domains in direct DNA breakage/repair and in chromosome aberration formation after mutagen treatment indicates the compatibility of the results

Sequence organization and evolutionary dynamics of Brachypodium-specific centromere retrotransposons

Citation: Qi, L., . . . Gill, B. (2013). Sequence organization and evolutionary dynamics of Brachypodium-specific centromere retrotransposons. Chromosome Research, 21, 507-521. https://doi.org/10.1007/s10577-013-9378-4Brachypodium distachyon is a wild annual grass belonging to the Pooideae, more closely related to wheat, barley, and forage grasses than rice and maize. As an experimental model, the completed genome sequence of B. distachyon provides a unique opportunity to study centromere evolution during the speciation of grasses. Centromeric satellite sequences have been identified in B. distachyon, but little is known about centromeric retrotransposons in this species. In the present study, bacterial artificial chromosome (BAC)-fluorescence in situ hybridization was conducted in maize, rice, barley, wheat, and rye using B. distachyon (Bd) centromere-specific BAC clones. Eight Bd centromeric BAC clones gave no detectable fluorescence in situ hybridization (FISH) signals on the chromosomes of rice and maize, and three of them also did not yield any FISH signals in barley, wheat, and rye. In addition, four of five Triticeae centromeric BAC clones did not hybridize to the B. distachyon centromeres, implying certain unique features of Brachypodium centromeres. Analysis of Brachypodium centromeric BAC sequences identified a long terminal repeat (LTR)-centromere retrotransposon of B. distachyon (CRBd1). This element was found in high copy number accounting for 1.6 % of the B. distachyon genome, and is enriched in Brachypodium centromeric regions. CRBd1 accumulated in active centromeres, but was lost from inactive ones. The LTR of CRBd1 appears to be specific to B. distachyon centromeres. These results reveal different evolutionary events of this retrotransposon family across grass species

A DNA mismatch repair gene links to the Ph2 locus in wheat

DNA mismatch repair is an essential system for maintaining genetic stability in bacteria and higher eukaryotes. Based on the conserved regions of the bacterial MutS gene and its homologues in yeast and human, a wheat cDNA homologue of MSH6, designated TaMSH7, was isolated by RT–PCR. The deduced amino acid sequence of TaMSH7 shows conserved domains characteristic of other MSH6 genes, with highest similarity to maize MSH7 and Arabidopsis MSH7. TaMSH7 is expressed in meristem tissue associated with a high level of mitotic and meiotic activity, with maximum expression in the reproductive organs of young flower spikes. TaMSH7 is located on the short arms of chromosomes 3A, 3B, and 3D and has been mapped within barley chromosome 3HS. The copy on 3DS is located within the region deleted in the wheat mutant ph2a, which shows altered recombination frequency in the interspecific hybrids. The relationship between the ph2a mutant and TaMSH7 gene function is discussed.Chongmei Dong, Ryan Whitford, and Peter Langridg

Chromosome painting in plants.

The current 'state-of-art' as to chromosome painting in plants is reviewed. We define different situations described as painting so far: i) Genomic in situ hybridisation (GISH) with total genomic DNA to distinguish alien chromosomes on the basis of divergent dispersed repeats, ii) 'Chromosomal in situ suppression' (CISS) hybridisation with chromosome-derived DNA probes and blocking of interchromosomally dispersed repeats by total genomic or C0 t-1 DNA in excess, iii) exceptional cases of single chromosome painting by probes containing chromosome-specific dispersed repeats, and iv) Fluorescence in situ hybridisation (FISH) with extended contigs of large insert clones for painting of those chromosomes of a euploid complement which harbour the cloned sequences. While GISH was successfully applied in most plant hybrids and/or their derivatives, painting of individual chromosomes by CISS hybridisations of chromosome-specific DNA probes have so far not revealed convincing results in plants. The reason for this failure and the use of possible alternative approaches are discussed. At least for small plant genomes, painting by large insert single sequence clones provides a promising alternative tool to solve cytogenetic questions, which up to now could not be tackled otherwise. An example of such a painting is described in detail for Arabidopsis thaliana