Application of fiber-FISH in physical mapping of Arabidopsis thaliana
Application of fiber-FISH in physical mapping of Arabidopsis thaliana
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DOI：
10.1139/gen-41-4-566
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发表时间：
1998-08-01
期刊：
影响因子：
3.1
通讯作者：
Jiang, JM
中科院分区：
文献类型：
--
作者：
Jackson, SA;Wang, ML;Jiang, JM
拟南芥因其基因组小、幼苗期短而成为遗传学研究的模式植物。然而，该物种的小染色体不适合进行经典的细胞遗传学研究。在这里，我们证明了使用延伸的DNA纤维的荧光原位杂交(FISH)技术可以成为对拟南芥基因组进行物理定位的有力工具。使用一种精细的纤维-FISH技术，我们能够测量长达1.71Mb的DNA簇，超过拟南芥基因组的1%。几个小的DNA基因座，包括端粒和一个分散的重复DNA序列mi167，也被用这种技术进行了分析。结果表明，如果没有已知的相邻DNA标记，这样的小DNA基因座就不能用纤维FISH精确定位。通过重叠群组装进行物理作图最困难的障碍之一是弥合相邻重叠群之间存在的间隙。目前可用的分子技术不足以准确估计这些缺口的物理大小。我们在拟南芥II号染色体物理重叠群图上分离到与第2和第3段相邻的细菌人工染色体(BAC)克隆，并将这些BAC克隆用于FISH分析，估计这两个缺口的物理大小分别为31kb和500kb以上。因此，我们已经证明了Fibre-FlSH是一种确定分子重叠群图上间隙的物理大小的有效技术。
Arabidopsis thaliana has become a model plant species for genetic studies because of its small genome and short juvenility period. However, the small chromosomes of this species are not suitable for classical cytogenetic studies. Here we demonstrate that the fluorescence in situ hybridization (FISH) technique using extended DNA fibers can be a powerful tool in the physical mapping of the A. thaliana genome. Using a refined fiber-FISH technique we were able to measure DNA clusters as long as 1.71 Mb, more than 1% of the A. thaliana genome. Several small DNA loci, including the telomeres and a dispersed repetitive DNA sequence, mi167, were also analyzed with this technique. The results show that without known adjacent DNA markers such small DNA loci cannot be mapped precisely using fiber-FISH. One of the most difficult obstacles in physical mapping by contig assembly is closing the gaps that are present between adjacent contigs. Currently available molecular techniques are not sufficient to accurately estimate the physical sizes of these gaps. We isolated bacterial artificial chromosome (BAC) clones bordering Saps 2 and 3 on the physical contig map of A. thaliana chromosome II. The BAC clones were used in fiber-FISH analysis and the physical sizes of the two gaps were estimated as 31 kb and more than 500 kb, respectively. Thus, we have demonstrated that fiber-FlSH is an efficient technique for determining the physical size of gaps on molecular contig maps.