S基因在甘蓝EDFs上的高分辨率荧光原位杂交定位

植物细胞壁及细胞质的存在和植物染色体所具有的高浓缩特性，限制高效率原位杂交定位在植物细胞内的进行。针对小型染色体芸薹属植物采用常规方法DNA制备纤维的效果不佳的特点，新建制备方法：利用减数分裂前期的染色体为材料，在硅化的玻片上先后通过蛋白酶解和乙醇：乙酸（3：1）的适当处理，采用移动界而法制备EDFs。制备的EDFs比未经伸长处理的染色体在经向和横向方面分别取得较高程度的伸长与膨胀，长度可达到89-257μm，比相应地中期染色体增长30107倍，分辨率可达42．853．0kb。利用SRK和SCR两种探针同时在甘蓝粗线期染色体和EDFs上进行了原位杂交，首次鉴定了S基因座在其单倍体基因组中单拷贝性。在杂交信号检测中尽管未经过信号放大，但仍然可以观察到清晰的绿色信号；经荧光显微镜观察，在单一的EDF上发现两个相距1μm的SCR和SRK的信号点，由此得出局部分辨率为4kb的最高伸长度。

Localization of S Genes on Extended DNA Fibers (EDFs) in Brassica oleracea by High-Resolution FISH

The compactness of plant chromosome and the structures of plant cell wall and cytoplasm pose a great resistance to fluorescence in situ hybridization (FISH), and consequently many new methods for improving spatial resolution are being exploited to overcome these problems. However, for plants with small chromosomes like rice and Brassica, there are still many difficulties.In this article a new and effective technique for preparation of extended DNA fibers (EDFs), using a series of treatments to prophase I chromosomes of Brassica oleracea PMCs, is presented. This technique allows longitudinal extension of the chromosomes 30-107 times longer than those of their metaphase counterparts. The length of the extended DNA fibers is between 89 μm and 273 μm,and the space resolution is 42.8-53.0 kb. Stretching ratios were assessed in a number of FISH experiments with super-stretched chromosomes from meiotic prophase I nuclei ofB. olerecea. Through FISH to EDFs ofpachytene chromosomes hybridized in situ with SRK (S-locus receptor kinase) and SP Ⅱ (S-locus protein Ⅱ)probes, for the first time we localized the accurate positions of S-locus and quantitatively analyzed the features of S genes in B. oleracea genome to show all S genes were single-copied. In addition, the length between two linked genes was measured to be about one micron. As a result, the highest space resolution which was about 4 kb was obtained.