The DNA hypomethylation effect of 5-azacytine (5-AC; a cytosine analog) is widely known. This agent has been used for rRNA gene expression studies of Triticeae amphiploids and hybrids regarding rye rRNA genes suppression caused by the wheat nucleolar dominance phenomenon. However, this situation is reverted by 5-AC treatment which activates rye rRNA gene expression as it has been intensively observed in triticale. For nucleolar dominance studies, we produced F1 multigeneric hybrids (AABBRHch; 2n = 6x = 42) from crosses between the triticale cultivar ‘Corgo’ (AABBRR; 2n = 6x = 42) and the tritordeum cultivars HT9 and HT31 (AABBHchHch; 2n = 6x = 42). The hybrid seeds were germinated in a low concentration of 5-AC (treatment) and in distilled water (nontreated control plants). Silver nitrate staining performed in one 5-AC-treated F1 hybrid revealed a reduced number of interphase cells with seven nucleoli, metaphases with eight Ag-NORs, and neocentromeres in the long arm of three wheat chromosomes. Nontreated hybrids presented six Ag-NORs per mitotic metaphase cell and a maximum of six nucleoli per interphase because of the 1R Ag-NOR suppression. No neocentromere was found in the control F1 hybrid plants. Both treated and nontreated seedlings were subsequently evaluated by fluorescent in situ hybridization performed with genomic and repetitive DNA probes to identify Hch and rye genomes, to confirm Ag-NORs location, and to detect inactive rDNA loci. DAPI counterstaining was also helpful for the detection of neocentromeres in the long arm of three wheat chromosomes. This study allowed us to suggest that 5-AC treatment specifically induced wheat neocentromeres in the F1 multigeneric triticale × tritordeum hybrids.
5-Azacytidine FISH Neocentromeres NOR Triticum aestivum L.
fluorescent in situ hybridization
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This work was supported by the POCTI/35107/AGR/2000 and PTDC/AGR-GPL/65876/2006 projects and the Ph.D. grant SFRH/BD/17348/2004 financed by the Portuguese Foundation for Science and the Technology (FCT).
Bajer A, Östergren G. Centromere-like behaviour of noncentromeric bodies. I. Neo-centric activity in chromosomal arms at mitosis. Hereditas 1968;47:563–98.Google Scholar
Choo KHA. Centromere DNA dynamics: latent centromeres and neocentromere formation. Am J Hum Genet 1997;61:1225–33.PubMedCrossRefGoogle Scholar
Dawe RK, Hiatt EN. Plant neocentromeres: fast, focused, and driven. Chromosom Res 2004;12:655–69.CrossRefGoogle Scholar
De Las Heras JI, King IP, Parker JS. 5-azacytidine induces chromosomal breakage in the root tips of wheat carrying the cuckoo chromosome 4S(L) from Aegilops sharonensis. Heredity 2001;87(4):474–9.PubMedCrossRefGoogle Scholar
Gerlach WL, Bedbrook JR. Cloning and characterization of ribosomal RNA genes from wheat and barley. Nucl Acids Res 1979;7:1869–85.PubMedCrossRefGoogle Scholar
Kakutani T, Jeddeloh JA, Flowers SK, et al. Developmental abnormalities and epimutations associated with DNA hypomethylation mutations. Proc Natl Acad Sci U S A 1996;93:12406–11.PubMedCrossRefGoogle Scholar
Lima-Brito J, Guedes-Pinto H, Harrison GE, Heslop-Harrison JS. Chromosome identification and nuclear architecture in triticale x tritordeum F1 hybrids. J Exp Bot 1996;47:583–8.CrossRefGoogle Scholar
Lima-Brito J, Guedes-Pinto H, Heslop-Harrison JS. The activity of nucleolar organizing chromosomes in multigeneric F1 hybrids involving wheat, triticale and tritordeum. Genome 1998;41:763–8.CrossRefGoogle Scholar
Maggert KA, Karpen GH. The activation of a neocentromere in Drosophila requires proximity to an endogenous centromere. Genetics 2001;158:1615–28.PubMedGoogle Scholar
Manzanero S, Puertas MJ. Rye terminal neocentromeres: characterisation of the underlying DNA and chromatin structure. Chromosoma 2003;111:408–15.PubMedCrossRefGoogle Scholar
Manzanero S, Puertas MJ, Jimenez G, Vega JM. Neocentric activity of rye 5RL chromosome in wheat. Chromosom Res 2000;8:543–54.CrossRefGoogle Scholar
Manzanero S, Vega JM, Houben A, Puertas MJ. Characterization of the constriction with the neocentric activity of 5RL chromosome in wheat. Chromosoma 2002;111:228–35.PubMedCrossRefGoogle Scholar
Nasuda S, Hudakova S, Schubert I, et al. Stable barley chromosomes without centromeric repeats. Proc Natl Acad Sci U S A 2005;102(28):9842–47.PubMedCrossRefGoogle Scholar
Neves N, Castilho A, Silva M, et al. Genomic interactions: gene expression, DNA methylation and nuclear architecture. Chromosomes Today 1997;12:182–200.Google Scholar
Neves N, Heslop-Harrison JS, Viegas W. rRNA gene activity and control of expression mediated by methylation and imprinting during embryo development in wheat x rye hybrids. Theor Appl Genet 1995;91:529–33.CrossRefGoogle Scholar
Ozkan H, Levy AA, Feldman M. Allopolyploidy-induced rapid genome evolution in the wheat (Aegilops–Triticum) group. Plant Cell 2001;13:1735–47.PubMedCrossRefGoogle Scholar
Rodriguez MJ, Lopez MA, Garcia-Orad A, Vig BK. Sequence of centromere separation: effect of 5-azacytidine-induced epigenetic alteration. Mutagenesis 2001;16(2):109–14.PubMedCrossRefGoogle Scholar
Stack S, Herikhoff L, Sherman J, Anderson L. Staining plant cells with silver. I. The salt nylon technique. Biotech Histochem 1991;1:69–78.PubMedCrossRefGoogle Scholar
Vanyushin BF, Shorning BY, Seredina AV, Aleksandrushkina NI. The effects of phytohormones and 5-azacytidine on apoptosis in etiolated wheat seedlings. Russ J Plant Physiol 2002;49(4):501–6.CrossRefGoogle Scholar
Vieira R, Queiroz A, Morais L, et al. 1R chromosome nucleolus organizer region activation by 5-azacytidine in wheat x rye hybrids. Genome 1990;33:707–12.Google Scholar