Non-denaturing fluorescence in situ hybridization: a transformative tool for chromosome identification and engineering for crop improvement in the plant genomics era.

This review highlights ND-FISH mechanisms, genomic integration, and workflows for karyotyping, rearrangement detection, and introgression, bridging cytogenetics and breeding for precision crop improvement. Fluorescence in situ hybridization (FISH) has been a pivotal technique for chromosome identification in plant species for over three decades. In particular, the non-denaturing FISH (ND-FISH) method, developed in 2009 and based on synthetic oligonucleotide probes derived from simple sequence repeats (SSRs), offers a highly efficient and labor-saving alternative to conventional FISH protocols. The ND-FISH method enables large-scale karyotyping at low cost, making it suitable for both large and small genomes, especially in polyploid plant species. In recent decades, improvements in chromosome preparation have facilitated high-throughput molecular cytogenetic identification for studying plant genetic variation and diversity. Notably, the rapid expansion of plant genomic resources and the development of bioinformatics-based computational tools have enabled the production of various types of diversified oligonucleotide probes. These advances support molecular cytogenetic mapping and precise chromosome engineering, as well as validation of genome assembly, which effectively bridges the gap between laboratory genomic research and practical field breeding applications. This review summarizes key technical advances and mechanistic insights into ND-FISH, highlights recent achievements, and discusses the prospects for its applications in the plant genomics era.