|Mcgrath, J Mitchell - Mitch|
Submitted to: Genome
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/23/2001
Publication Date: N/A
Interpretive Summary: Visualizing chromosomes through the microscope is among the earliest methods applied in plant biology, and tremendous advances in plant improvement have been made using the knowledge gained from cytogenetics. Visualizing individual genes and other features of chromosomes has been much more difficult, and has been possible only with the development of fluorescently tagged markers that can be superimposed on the entire chromosome. A method was developed to apply this technique of Fluorescent In Situ Hybridization (FISH) to plants with small chromosomes, such as potato. Further, the method allows screening chromosomes with multiple markers sequentially so that important features can be examined using the same, difficult to obtain, chromosome preparations. The impact of this new technique allows plant cytogeneticists to precisely locate individual genes on chromosomes relative to other genes and provides an additional tool for plant breeders to determine the genetic basis of agronomic traits
Technical Abstract: Genomic in situ hybridization (GISH) is one of the most popular and effective techniques for detecting alien chromatin introgressed into breeding lines; however, GISH analysis alone does not reveal the genetic identify of the alien chromosomes. We previously isolated a set of bacterial artificial chromosomes (BACs) specific to each of the 12 potato chromosomes. These BAC clones can be used as chromosome-specific cytogenic DNA markers (CSCDMs) for potato chromosome identification. Here we demonstrate that GISH and fluorescence in situ hybridization (FISH), using CSCDMs, can be performed sequentially on the same chromosome preparations. Somatic metaphase chromosomes prepared using an enzymatic digestion and "flame-drying" procedure allows repeated probing up to five times without significant damage to chromosome morphology. The sequential GISH and FISH analyses reveal the genomic origin and genetic identity of the alien chromosomes in a single experiment and also determine whether an alien chromosome has been added to the genetic background of potato or is substituting for a homeologous potato chromosome. The sequential GISH and FISH procedures should be widely applicable for germplasm characterization, especially in plant species with small-sized chromosomes.