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Detection of single‐copy genes and chromosome rearrangements in Petunia hybrida by fluorescence in situ hybridization
Author(s) -
Fransz Paul F.,
Stam Maike,
Montijn Bob,
Hoopen Rogier Ten,
Wiegant Joop,
Kooter Jan M.,
Oud Oof,
Nanninga Nanne
Publication year - 1996
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1046/j.1365-313x.1996.9050767.x
Subject(s) - biology , microbiology and biotechnology , fluorescence in situ hybridization , chromosome , gene , genetics , hybridization probe , in situ hybridization , gene expression
Summary DNA sequences homologous to single‐copy genes were labelled with biotinylated dUTP or digoxygenin‐labelled dUTP and hybridized to chromosome spreads. The hybridization signals were visualized with fluorescent avidin‐ or antibody‐conjugates. This method allowed the detection of DNA targets on metaphase chromosomes as small as 1.4 kb. The hybridization signals were identified as fluorescent spots on both sister chromatids. Using an 18S rDNA probe as marker to identify chromosomes II and III it was possible to assign single‐copy genes to these chromosomes. In the line V30 the endogenous chalcone synthase gene ( chsA ) was mapped at the distal end of the short arm of chromosome 5. The cDNA probe for this single‐copy gene was 1.4 kb. In contrast, in the lines Mitchell and V26 chsA was localized at the distal end of the long arm of chromosome 3, suggesting that a chromosomal rearrangement had taken place. In a transformed Petunia uidA , transgenes were detected using a 2.7 kb probe. One transgene was mapped on one of the homologues of chromosome II proximal to the ribosomal genes. This homologue could be distinguished from the other by having the ribosomal genes at the distal end of the long arm. Using multicolour fluorescence in situ hybridization it was shown that it is possible to detect the endogenous chsA genes and both transgenes simultaneously.