Improvement of high-resolution fluorescence in situ hybridisation mapping on chromosomes of Brassica oleracea var. capitata
Improvement of high-resolution fluorescence in situ hybridisation mapping on chromosomes of Brassica oleracea var. capitata
复制标题
甘蓝染色体高分辨率荧光原位杂交作图的改进。
作者：
Yang K.;Zhang Y.;Converse R.;Lv J.;Shi M.;Zhang H.;Zhu L.
基于染色体的原位杂交（FISH）作图的低分辨率主要是由于植物细胞壁和细胞质的结构以及规则染色体的紧凑性，这代表了FISH的显著障碍。为了提高空间分辨率和信号检测灵敏度，我们提供了一种可重复的方法来生成高质量的延伸染色体，其长度是粗线期对应物的约13倍。我们证明，蛋白酶K在此过程中使用的是至关重要的拉伸粗线期染色体ofBrassica oleracein的背景下，一个修改的卡诺氏II固定液（6：1：3，乙醇：氯仿：乙酸）。在几个FISH实验中评估了超拉伸染色体的质量。来自超拉伸染色体上的重复5S rDNA和单拷贝ARC 1的FISH信号比其他不同类型的染色体上的信号更亮，这是由于对拉伸粗线期染色体上的靶标的可及性增强。总之，所得到的延伸染色体适用于重复DNA序列的FISH定位和单拷贝基因座的定位，并且在超拉伸染色体上进行的FISH可以实现比其他基于染色体的FISH定位技术显著更高的灵敏度和空间分辨率。
The low resolution of chromosome‐based Fluorescencein situhybridisation (FISH) mapping is primarily due to the structure of the plant cell wall and cytoplasm and the compactness of regular chromosomes, which represent a significant obstacle to FISH. In order to improve spatial resolution and signal detection sensitivity, we provide a reproducible method to generate high‐quality extended chromosomes that are ~13 times as long as their pachytene counterparts. We demonstrate that proteinase K used in this procedure is crucial for stretching pachytene chromosomes ofBrassica oleraceain the context of a modified Carnoy's II fixative (6:1:3, ethanol:chloroform:acetic acid). The quality of super‐stretched chromosomes was assessed in several FISH experiments. FISH signals from both repetitive 5S rDNA and single‐copyARC1on super‐stretched chromosomes are brighter than those on other different types of chromosome due to enhanced accessibility to targets on stretched pachytene chromosomes. In conclusion, the resulting extended chromosomes are suitable for FISH mapping for repetitive DNA sequences and the localisation of a single‐copy locus, and FISH performed on super‐stretched chromosomes can achieve significantly higher sensitivity and spatial resolution than other chromosome‐based FISH mapping techniques.