Systematic Application of DNA Fiber-FISH Technique in Cotton

<div><p>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, <i>G. hirsutum</i>, <i>G. herbaceum</i> and <i>G. arboreum</i>. However, most of the telomeres (>91.0%) were below 10 kb. The length of 5S rDNA was revealed as 964 kb in <i>G. herbaceum</i> whereas, in <i>G. arboreum</i>, 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.</p></div

Telomere-size distributions in <i>G. hirsutum</i>, <i>G. herbaceum</i> and <i>G. arboreum</i>.

<p>Two hundred telomere signals for <i>G. hirsutum</i> (A), <i>G. herbaceum</i> (B) and <i>G. arboreum</i> (C) were measured from three individual fiber-FISH slides. The percentage of telomere within different size-rang were indicated. The sizes of the longest and shortest telomeres were showed on the corresponding measurements.</p

Evaluation of the resolution of extended fiber and molecule by two BAC clones.

a<p>the 7.5-kb vector was included.</p

FISH on extended genomic DNA fibers and BAC molecules.

<p>A) FISH signals derived from two BAC clones 173C03 (green) and 174A01 (red) on extended genomic fibers of <i>G. hirsutum</i>. B) FISH mapping of BAC 173C03 molecules. Both clones 174A01 (red) and BAC 173C03 (green) were used as probe and hybridized onto 173C03 molecules to visualize the BAC 173C03 molecules and overlap regions. Red signals (174A01) covered most parts of BAC 173C03 molecules indicating that most parts of 173C03 were overlapped with 174A01. C) FISH mapping of BAC 174A01 molecules. Probes 174A01 (green) and BAC 173C03 (red) were hybridized onto 174A01 molecules. To facilitate the identification of overlapped regions, BAC vector (red, arrow) was labeled and hybridized simultaneously with two BAC probes. Bars are 10 µm.</p

List of primers.

<p>List of primers.</p

Mitotic metaphase chromosomes of Japanese foxtail.

<p>Scale bar is 10 µm.</p

Flow cytometric analysis of homogenates prepared from perennial ryegrass (a) and Japanese foxtail (b).

<p>Flow cytometric analysis of homogenates prepared from perennial ryegrass (a) and Japanese foxtail (b).</p

Genomic southern blot hybridization analysis of Japanese foxtail digested with <i>Eco</i>RI, <i>Hae</i>III and both enzymes.

<p>M, DIG labeled DNA molecular weight.</p