Systematic application of DNA fiber-FISH technique in cotton.

ABSTRACT

Fluorescence in situ hybridization on extended DNA (fiber-FISH) is a powerful tool in high-resolution physical mapping. To introduce this technique into cotton, we developed the technique and tested it by deliberately mapping of telomere and 5S rDNA. Results showed that telomere-length ranged from 0.80 kb to 37.86 kb in three species, G. hirsutum, G. herbaceum and G. arboreum. However, most of the telomeres (>91.0%) were below 10 kb. The length of 5S rDNA was revealed as 964 kb in G. herbaceum whereas, in G. arboreum, it was approximately three times longer (3.1 Mb). A fiber-FISH based immunofluorescence method was also described to assay the DNA methylation. Using this technique, we revealed that both telomere and 5S rDNA were methylated at different levels. In addition, we developed a BAC molecule-based fiber-FISH technique. Using this technique, we can precisely map BAC clones on each other and evaluated the size and location of overlapped regions. The development and application of fiber-FISH technique will facilitate high-resolution physical mapping and further directed sequencing projects for cotton.