Fluorescence in situ hybridization of single copy transgenes in rice chromosomes

Fluorescence in situ hybridization (FISH) is a powerful tool for visualizin g the chromosomal location of targeted sequences and has been applied in ma ny areas, including karyotyping, breeding and characterization of genes int roduced into the plant genome. A simple, routine and sensitive FISH procedu re was developed for localizing single copy genes in rice (Oryza sativa L.) metaphase chromosomes. We used digoxygenin-labeled endogenous or T-DNA seq uences as small as 5.6 kb to probe corresponding endogenous sequences or th e T-DNA insert in denatured rice metaphase chromosomes prepared from root m eristem tissue. The hybridized probe sequence was labeled with cy3-conjugat ed anti-mouse IgG and visualized using fluorescence microscopy. Single copy and multiple copy introduced T-DNA sequences, as well as endogenous sequen ces, were localized on the chromosomes. The FISH protocol was effectively u sed to screen the chromosomal location of introduced T-DNA and number of in tegration loci in rice.