National Key Laboratory of Crop Genetics and Germplasm Enhancement, Cotton Research Institute, Nanjing Agricultural University, Nanjing, China.
PLoS One. 2013 Sep 27;8(9):e75674. doi: 10.1371/journal.pone.0075674. eCollection 2013.
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.
荧光原位杂交（FISH）在扩展 DNA（纤维 FISH）上是一种用于高分辨率物理作图的强大工具。为了将该技术引入棉花中，我们开发了该技术，并通过故意对端粒和 5S rDNA 作图进行了测试。结果表明，在三个物种（陆地棉、草棉和亚洲棉）中，端粒长度范围从 0.80 kb 到 37.86 kb。然而，大多数端粒（>91.0%）都小于 10 kb。在草棉中，5S rDNA 的长度为 964 kb，而在亚洲棉中，其长度大约是三倍（3.1 Mb）。还描述了一种基于纤维 FISH 的免疫荧光法来检测 DNA 甲基化。使用该技术，我们揭示了端粒和 5S rDNA 都在不同水平上被甲基化。此外，我们还开发了基于 BAC 分子的纤维 FISH 技术。使用该技术，我们可以精确地将 BAC 克隆彼此作图，并评估重叠区域的大小和位置。纤维 FISH 技术的开发和应用将有助于棉花的高分辨率物理作图和进一步的定向测序项目。
