Higher axial-resolution and sensitivity pachytene fluorescence in situ hybridization protocol in tetraploid cotton
Higher axial-resolution and sensitivity pachytene fluorescence in situ hybridization protocol in tetraploid cotton
复制标题
DOI：
10.1007/s10577-009-9085-3
复制
发表时间：
2009-10
影响因子：
2.6
通讯作者：
Kai Wang;Zaijie Yang;C. Shu;Jing Hu;Qiuyun Lin;Wen-pan Zhang;Wangzhen Guo;Tianzhen Zhang
中科院分区：
文献类型：
--
作者：
Kai Wang;Zaijie Yang;C. Shu;Jing Hu;Qiuyun Lin;Wen-pan Zhang;Wangzhen Guo;Tianzhen Zhang
基于粗线期染色体的荧光原位杂交(FISH)已成为构建高轴向分辨率和高灵敏度细胞遗传学图谱的重要细胞遗传学工具。然而，由于染色体制备的困难，这项技术在棉花上的应用一直比较滞后。到目前为止，基于棉花粗线期染色体的FISH尚未见报道。本研究首次提出了四倍体棉花粗线期染色体制备方法。该方法产生了适合于大小DNA探针FISH标记的染色体铺展。系统分析了FISH在有丝分裂中期和粗线期染色体上的两个重要参数：轴向分辨率和敏感度。结果表明，粗线虫的DNA靶和小至3kb的低拷贝靶分别被分辨和检测到。我们的FISH方案的应用将继续改进，并为构建棉花整合的高轴向分辨率细胞遗传学图谱提供一个起点。
Fluorescence in situ hybridization (FISH) based on pachytene chromosomes has become an important cytogenetic tool to construct high axial-resolution and sensitivity cytogenetic maps. However, the application of this technique in cotton has lagged behind due to difficulties in chromosome preparation. To date, successful FISH based on cotton pachytene chromosomes has not been reported. In this study, the first protocol developed for pachytene chromosome preparation in tetraploid cotton is presented. This protocol yielded chromosome spreads suitable for large and small DNA probe FISH labeling. Two important parameters, axial-resolution and sensitivity, of FISH on mitotic metaphase and pachytene chromosomes were systematically analyzed. The results demonstrated that DNA targets separated by 0.6 cM and low-copy targets as small as 3-kb were resolved and detected, respectively, in pachytene FISH. The application of our FISH protocol will continue to improve and provide a point of departure for constructing an integrated high axial-resolution cytogenetic map in cotton.