High-density fluorescence in situ hybridization signal detection on barley (Hordeum vulgare L.) chromosomes with improved probe screening and reprobing procedures

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Abstract:

The barley (Hordeum vulgare L.) genome was screened to identify sequences that could be used for fluorescence in situ hybridization (FISH). From 2000 transformed bacterium colonies carrying barley clones, 56 colonies were selected on the basis of the patterns that their PCR products produced when subjected to agarose gel electrophoresis. Among them, 42 (75%) exhibited fluorescent signals on barley chromosomes after in situ hybridization using the directly labeled PCR products. Sequencing revealed seven clones, pHv-365, pHv-177, pHv-1112, pHv-689, pHv-1476, pHv-1889, and pHv-1972, to be newly identified FISH-positive sequences. The remainder possess previously described sequences such as 5S, GAA microsatellite, centromere repeats, HVT01, and pHvMWG2315 (324 bp repeat). It is shown here that a combination of five probes, which produce strong signals on barley chromosomes, pHv-38 (5S), pHv-365, pHv-961 (HVT01), GAA, and TAG microsatellites, offer unequivocal recognition of each chromosome. The combination of three probes, i.e., pHv-1123 (barley 324 bp repeat), GAA, and TAG, decorated entire chromosomes with fine dotted signals and was useful for detecting the break points of aberrant chromosomes. The signals’ distributions of pHv-177, pHv-1112, and TAG were highly polymorphic. An improved reprobing procedure and its usefulness are also discussed.

Le génome de l’orge (Hordeum vulgare L.) a été criblé pour identifier des séquences pouvant servir en hybridation in situ en fluorescence. À partir de 2000 colonies bactériennes transformées contenant des clones d’orge, 56 colonies ont été retenues en raison de leur amplicon PCR suite à une électrophorèse sur gel d’agarose. Parmi ceux-ci, 42 (75 %) présentaient des signaux fluorescents sur des chromosomes d’orge après hybridation in situ à l’aide des produits PCR marqués. Le séquençage a montré que sept clones, pHv-365, pHv-177, pHv-1112, pHv-689, pHv-1476, pHv-1889 et pHv-1972, constituaient de nouvelles sondes FISH. Les autres clones contenaient diverses séquences déjà décrites comme l’ADNr 5S, le microsatellite GAA, des répétitions centromériques, HVT01, pHvMWG2315 (répétition de 324 pb). Les auteurs montrent qu’une combinaison de cinq sondes produisant de forts signaux d’hybridation sur les chromosomes de l’orge, pHv-38 (5S), pHv-365, pHv-961 (HVT01) ainsi que les microsatellites GAA et TAG, permettent d’identifier clairement chacun des chromosomes. La combinaison de trois sondes, soit pHv-1123 (répétition de 324 pb), GAA et TAG, permet de décorer les chromosomes entiers d’un signal ponctuel fin et d’ainsi détecter les cassures chez les chromosomes aberrants. La distribution des signaux obtenus avec les sondes pHv-177, pHv-1112 et TAG était très polymorphe. Les auteurs présentent également une méthode améliorée de re-hybridation des lames et discutent de l’utilité de celle-ci.

Document Type: Research Article

Publication date: February 1, 2011

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  • From its inception in 1957, this international cytogenetics journal has catered to the research areas of the members of the Genetics Society of Canada; traditionally, these have included agriculture, entomology, genetics/cytogenetics, and evolutionary mechanisms. The contents of the journal have evolved as contributors developed new technologies and interests. A 20-member Editorial Board is composed of scientists from around the world. Reviews and commentary from respected experts are often featured.
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