In situ hybridization of biotin-labeled probes to plant chromosomes is a powerful technique enabling the physical mapping of DNA sequences and the “tagging” of chromosomes for identification ( 1 ). The method described here is based on Rayburn and Gill ( 2 ) and is particularly successful for detection of sequences present in multiple copies. In this method the signal is detected using a streptavidin/horse-radish peroxidase conjugate, which binds to the biotin-labeled probe, and the color reaction is produced by the addition of hydrogen peroxide and the dye, 3,3′-diaminobenzidine tetrahydrochloride dihyhdrate (DAB). The signal is seen as a dark precipitate (Fig. 1 ) that maintains color intensity for several years, provided slides are kept away from bright light. Alternative methods of probing and detection are given in chapter 26 and chapter 27 . The whole procedure can be run in 1 d, with a 4-h hybridization period, but we prefer to begin the method in the afternoon and to hybridize overnight.   Fig. 1.  In situ hybridization using biotin-labeled probes. Signal detection was achieved using a streptavidin-horseradish peroxidase conjugate, which binds to the biotin followed by addition of hydrogen peroxide and the dye 3,3′-diaminobenzidine tetrahydrochloride dihydrate (DAB). (A) Localization of ribosomal RNA genes in rye. Two sites are detected (arrows) corresponding with the nucleolar organizers on chromosome 1 (A. Karp, unpublished results). (B) Localization of a B-chromosome specific probe (arrows) to the two B chromosomes of a rye plant ( 3 ). (Scale bar = 10 �m).