Localization of Rad50, a single-copy gene, on group 5 chromosomes of wheat, using a FISH protocol employing tyramide for signal amplification (Tyr-FISH).
An indirect tyramide signal amplification FISH technique (Tyr-FISH) was adapted to detect single-copy genes in plants. In this study, Rad50 loci (responsible for a protein involved in the repair of breaks in double-stranded DNA) were localized on the chromosomes of homologous group 5 in common wheat (Triticum aestivum). Three loci, Rad50A, Rad50B and Rad50D, none of which have been previously mapped using any kind of molecular approach, were localized at similar positions on the short arm of chromosomes 5A, 5B and 5D, respectively. The loci are all proximal to 5S ribosomal gene clusters. The sensitivity and efficiency of the Tyr-FISH technique was tested using 5 differently sized fragments of the gene Rad50D as probes in FISH involving the chromosomes of the diploid wheat Triticum tauschii (the D-genome donor species of the hexaploid wheats). The procedure, which provided specific signal amplification with low level background, detected target DNA sequences as small as 2 kb with a frequency of 37.5%.
Pérez
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Department of Cell Biology and Genetics, University of Alcalá, Alcalá de Henares, Spain.
de Bustos
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Jouve
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Cuadrado
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D032461:Chromosomes, Plant / Q000235:genetics*; D015342:DNA Probes / Q000737:chemistry; D018744:DNA, Plant; D005456:Fluorescent Dyes / Q000378:metabolism; D018628:Gene Dosage; D017343:Genes, Plant / Q000235:genetics*; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D007211:Indoles / Q000378:metabolism; D021141:Nucleic Acid Amplification Techniques; D010940:Plant Proteins / Q000235:genetics*; D012091:Repetitive Sequences, Nucleic Acid; D017422:Sequence Analysis, DNA; D014908:Triticum / Q000235:genetics*; D014439:Tyramine / Q000031:analogs & derivatives
D015342:DNA Probes; D018744:DNA, Plant; D005456:Fluorescent Dyes; D007211:Indoles; D010940:Plant Proteins; C007293:DAPI; D014439:Tyramine