棉花gDNA体细胞染色体FISH技术

Protocol of Cotton FISH of Somatic Chromosomes with gDNA as Probes

介绍了棉花基因组ＤＮＡ（ｇｅｎｏｍｅＤＮＡ，简为ｇＤＮＡ）体细胞染色体荧光原位杂交〔ＦＩＳＨ〕的技术流程，并着重分析和讨论了影响试验结果的关键因素，包括染色体和探针的变性条件、染色体的蛋白酶Ｋ处理技巧等。试验中作为靶ＤＮＡ的体细胞染色体采用棉属异源四倍体种海岛棉；探针和封阻均采用ｇＤＮＡ，材料是棉属二倍体种Ａ染色体组（Ａｇｅｎｏｍｅ）的棉种（亚洲棉和草棉）和Ｄ染色体组（Ｄｇｅｎｏｍｅ）的棉种（瑟伯氏棉、雷蒙德氏棉、戴维逊氏棉等），分别交互使用。试验结果比较理想，获得良好的ＦＩＳＨ片子，而且重复性好。

Accessions used in the study included Gossypium barbadense as target DNA(somatic chromosomes), gDNA (genome DNA) of species of A genome including G.arboreum and G.herbaceum, of D genome including G.davidsonii, G.thurberi and G.raimondii, as probe DNA and/or block DNA, respectively. This paper described protocol of the FISH (Fluorescence in situ hybridization), with discussions emphasized on the key procedures of denaturation of chromosomes and probes, of proteinase K was used in treatment to chromosome preparations.