Department of Botany and Biodiversity Research, University of Vienna, Vienna, Austria.
Department of Biological Science, College of Bioscience and Biotechnology, Chungnam National University, Daejeon, South Korea.
Methods Mol Biol. 2023;2672:257-264. doi: 10.1007/978-1-0716-3226-0_16.
Fluorescence in situ hybridization allows for the mapping of various sequence types in the genomes and is thus widely used in structural, functional, and evolutionary studies. One particular type of in situ hybridization that specifically allows to map whole parental genomes in diploid and polyploid hybrids is genomic in situ hybridization (GISH). The efficiency of GISH, i.e., the specificity of hybridization of genomic DNA probes to the parental subgenomes in hybrids depends, among others, on the age of the polyploids and the similarity of the parental genomes, specifically their repetitive DNA fractions. Typically, high levels of overall repeat similarity between the parental genomes result in lower efficiency of GISH. Here, we present the formamide-free GISH (ff-GISH) protocol that can be applied to diploid and polyploid hybrids of both monocots and dicots. ff-GISH allows higher efficiency of the labeling of the putative parental genomes compared to the standard GISH protocol and allows discrimination of parental chromosome sets that share up to 80-90% repeat similarity. This modified method is nontoxic, is simple, and lends itself to modifications. It can also be used for standard FISH and mapping of individual sequence types in chromosomes/genomes.
荧光原位杂交允许在基因组中定位各种序列类型，因此广泛用于结构、功能和进化研究。一种特别允许在二倍体和多倍体杂种中定位整个亲本基因组的原位杂交方法是基因组原位杂交（GISH）。GISH 的效率，即基因组 DNA 探针与杂种中亲本亚基因组的杂交特异性，取决于多倍体的年龄和亲本基因组的相似性，特别是它们的重复 DNA 分数。通常，亲本基因组之间的整体重复相似性水平较高会导致 GISH 效率降低。在这里，我们介绍了无甲酰胺的 GISH（ff-GISH）方案，该方案可应用于单子叶植物和双子叶植物的二倍体和多倍体杂种。与标准 GISH 方案相比，ff-GISH 可以提高标记假定亲本基因组的效率，并允许区分具有高达 80-90%重复相似性的亲本染色体组。这种改良的方法是无毒的，简单易行，可以进行修改。它也可用于标准的 FISH 和染色体/基因组中单个序列类型的定位。
