Abstract
Brachypodium distachyon (Brachypodium) is now intensively utilized as a model grass species in various biological studies. Its favorable cytological features create a unique foundation for a convenient system in mutagenesis, thereby potentially enabling the ‘hot spots’ and ‘cold spots’ of DNA damage in its genome to be analyzed. The aim of this study was to analyze the involvement of 5S rDNA, 25S rDNA, the Arabidopsis-type (TTTAGGG)n telomeric sequence and the Brachypodium-originated centromeric BAC clone CB33J12 in the micronuclei formation in Brachypodium root tip cells that were subjected to the chemical clastogenic agent maleic hydrazide (MH). To the best of our knowledge, this is the first use of a multicolor fluorescence in situ hybridization (mFISH) with four different DNA probes being used simultaneously to study plant mutagenesis. A quantitative analysis allowed ten types of micronuclei, which were characterized by the presence or absence of specific FISH signal(s), to be distinguished, thus enabling some specific rules governing the composition of the MH-induced micronuclei with the majority of them originating from the terminal regions of chromosomes, to be identified. The application of rDNA sequences as probes showed that 5S rDNA-bearing chromosomes are involved in micronuclei formation more frequently than the 25S rDNA-bearing chromosomes. These findings demonstrate the promising potential of Brachypodium to be a useful model organism to analyze the effects of various genotoxic agents on the plant nuclear genome stability, especially when the complex FISH-based and chromosome-specific approaches such as chromosome barcoding and chromosome painting will be applied in future studies.
Highlights
Information regarding the distribution of DNA damage in plant chromosomes may be of great importance in understanding the biological impact of environmentally induced genotoxic effects
To the best of our knowledge, this is the first study on the susceptibility of Brachypodium to mutagenic treatment linked with fluorescence in situ hybridization (FISH)-based qualitative and quantitative analyses of the effects of such a treatment on the nuclear genome stability visualized at the cytomolecular level
Only 10% of maleic hydrazide (MH)-induced micronuclei with telomeric DNA sequences were reported in barley, though 46% of them had both telomeric and ribosomal DNA (rDNA) (5S or 25S) signals [5]. These results indicate that micronuclei in Brachypodium after MH treatment arise mostly from the terminal, acentric fragments of chromosomes, while in barley MH preferably leads to large acentric fragments including rDNA loci that are located in the interstitial parts of the chromosomes or near the centromere regions
Summary
Information regarding the distribution of DNA damage in plant chromosomes may be of great importance in understanding the biological impact of environmentally induced genotoxic effects. MFISH-Based Analysis of Micronuclei in Brachypodium distachyon breakage in plant chromosomes is possible using fluorescence in situ hybridization (FISH) [1, 2]. This technique provides the opportunity to detect even small aberrations in both dividing and non-dividing cells. The distribution of chromosome aberrations in such species has as yet only been analyzed using such probes, as ribosomal DNA (rDNA) and Arabidopsis thaliana (Arabidopsis)-type (TTTAGGG)n telomeric sequences. These probes usually have no chromosome specificity, they are relatively widely applied because of their evolutionarily conserved nature and ‘universal’ character [1, 6, 7]
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