Localization of S Genes on Extended DNA Fibers (EDFs) in Brassica oleracea by High-Resolution FISH

The compactness of plant chromosome and the structures of plant cell wall and cytoplasm pose a great resistance to fluorescence in situ hybridization (FISH), and consequently many new methods for improving spatial resolution are being exploited to overcome these problems. However, for plants with small chromosomes like rice and Brassica, there are still many difficulties. In this article a new and effective technique for preparation of extended DNA fibers (EDFs), using a series of treatments to prophase I chromosomes of Brassica oleracea PMCs, is presented. This technique allows longitudinal extension of the chromosomes 30–107 times longer than those of their metaphase counterparts. The length of the extended DNA fibers is between 89 um and 273 um, and the space resolution is 42.8–53.0 kb. Stretching ratios were assessed in a number of FISH experiments with super-stretched chromosomes from meiotic prophase I nuclei of B. olerecea. Through FISH to EDFs of pachytene chromosomes hybridized in situ with SRK (S-locus receptor kinase) and SPII (S-locus protein II)probes, for the first time we localized the accurate positions of S-locus and quantitatively analyzed the features of S genes in B. oleracea genome to show all S genes were single-copied. In addition, the length between two linked genes was measured to be about one micron. As a result, the highest space resolution which was about 4 kb was obtained.