ABSTRACT
Fluorescence in situ hybridization (FISH) is a molecular cytogenetic technique that allows localization of DNA sequences by hybridizing their probes on chromosomal targets. The results can be visualized under the microscope using a probe directly labeled with a fluorescent dye or a probe containing a hapten molecule that is further detected biochemically or with antibodies conjugated with fluorescent dye molecules. Initially, FISH was confined to localization studies of repetitive DNA sequences on highly condensed mitotic metaphase chromosomes and interphase nuclei (reviews in Trask, 1991; Jiang et al., 1996). However, for most detailed chromosome mapping studies, highly condensed metaphase complements are insufficient to distinguish neighbor DNA targets less than 1 to 2 Mb apart. Higher spatial resolution values of up to 50 to 100 kb can be achieved with FISH on interphase nuclei, but these targets lack the information of the native chromosomal structure. A better alternative for hybridizations in diploid plant species is the less contracted pachytene chromosomes, which generally measure 10 to 40 times longer than their mitotic metaphase counterparts and display a differentiated pattern of heterochromatin blocks (de Jong et al., 1999). Modern cytogenetic maps are mostly based on FISH position of genetically defined single-copy sequences on the pachytene chromosomes and are highly informative to support the construction of physical maps for map-based cloning projects and to position genes in pericentromere heterochromatin regions where recombination estimates are unreliable due to very low frequency of crossover events (Roberts, 1965; Zhong et al., 1999).
