The genetic identity of alien chromosomes in potato breeding lines revealed by sequential GISH and FISH analyses using chromosome-specific cytogenetic DNA markers

Genomic in situ hybridization (GISH) is one of the most popular and effecti ve techniques for detecting alien chromatin introgressed into breeding line s; however, GISH analysis alone does not reveal the genetic identity of the alien chromosomes. We previously isolated a set of bacterial artificial ch romosomes (BACs) specific to each of the 12 potato chromosomes. These BAC c lones can be used as chromosome-specific cytogenetic DNA markers (CSCDMs) f or potato chromosome identification. Here we demonstrate that GISH and fluo rescence in situ hybridization (FISH), using CSCDMs, can be performed seque ntially on the same chromosome preparations. Somatic metaphase chromosomes prepared using an enzymatic digestion and "flame-drying" procedure allows r epeated probing up to five times without significant damage to chromosome m orphology. The sequential GISH and FISH analyses reveal the genomic origin and genetic identity of the alien chromosomes in a single experiment and al so determine whether an alien chromosome has been added to the genetic back ground of potato or is substituting for a homoeologous potato chromosome. T he sequential GISH and FISH procedures should be widely applicable for germ plasm characterization, especially in plant species with small-sized chromo somes.