The low resolution of chromosome-based Fluorescence in situ hybridisation (FISH) mapping is primarily due to the structure of the plant cell wall and cytoplasm and the compactness of regular chromosomes, which represent a significant obstacle to FISH. In order to improve spatial resolution and signal detection sensitivity, we provide a reproducible method to generate high-quality extended chromosomes that are ~13 times as long as their pachytene counterparts. We demonstrate that proteinase K used in this procedure is crucial for stretching pachytene chromosomes of Brassica oleracea in the context of a modified Carnoy's II fixative (6:1:3, ethanol:chloroform:acetic acid). The quality of super-stretched chromosomes was assessed in several FISH experiments. FISH signals from both repetitive 5S rDNA and single-copy ARC1 on super-stretched chromosomes are brighter than those on other different types of chromosome due to enhanced accessibility to targets on stretched pachytene chromosomes. In conclusion, the resulting extended chromosomes are suitable for FISH mapping for repetitive DNA sequences and the localisation of a single-copy locus, and FISH performed on super-stretched chromosomes can achieve significantly higher sensitivity and spatial resolution than other chromosome-based FISH mapping techniques.
Keywords: Fluorescence in situ hybridisation; Pachytene; proteinase K; super-stretched chromosomes.
© 2015 German Botanical Society and The Royal Botanical Society of the Netherlands.