Systematic application of DNA fiber-FISH technique in cotton.
Systematic application of DNA fiber-FISH technique in cotton.
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DOI：
10.1371/journal.pone.0075674
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Zhang T
中科院分区：
文献类型：
--
作者：
Wang K;Zhang W;Jiang Y;Zhang T
扩展DNA荧光原位杂交技术（fiber-FISH）是高分辨率物理制图的有力工具。为了将该技术引入棉花，我们开发了该技术，并通过故意定位端粒和5S rDNA进行了测试。结果表明，毛毛、草本和木本三种植物的端粒长度在0.80 ~ 37.86 kb之间。然而，绝大多数（91.0%）端粒在10 kb以下。结果表明，草本植物的5S rDNA长度为964 kb，而木本植物的5S rDNA长度约为3.1 Mb，是草本植物的3倍。还描述了一种基于纤维- fish的免疫荧光方法来检测DNA甲基化。利用这种技术，我们发现端粒和5S rDNA都在不同水平上甲基化。此外，我们还开发了一种基于BAC分子的纤维- fish技术。利用该技术，我们可以精确地绘制BAC克隆之间的相互关系，并评估重叠区域的大小和位置。fiber-FISH技术的发展和应用将促进棉花的高分辨率物理制图和进一步定向测序项目。
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.
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