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

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 identity 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 cytogenetic 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 homoeologous potato chromosome. The sequential GISH and FISH procedures should be widely applicable for germplasm characterization, especially in plant species with small-sized chromosomes.

Genealogical relationships of southern Ontario polyploid unisexual salamanders (genus Ambystoma) inferred from intergenomic exchanges and major rDNA cytotypes

“…We used GISH to examine intergenomic exchanges in 110 unisexual individuals that included larvae and adults collected from 18 isolated populations. Although sequential and simultaneous GISH/FISH have been applied to reveal alien chromatin introgression and genetic identification of alien chromosomes in plants (Moscone et al 1996, Dong et al 2001, Kamstra et al 2004, Zheng et al 2006, no such experimentations have been attempted in any vertebrates that have a hybrid origin. For better resolution, we tentatively combined FISH-rDNA and GISH sequentially or simultaneously for most specimens to identify both genomic constitutions and major rDNA cytotypes on the same spreads.…”

Transfer of tuber soft rot and early blight resistances from Solanum brevidens into cultivated potato

“…This question would be difficult to address using a DNA marker-based approach due to the tetraploidy and high heterozygosity of the potato genome. Dong et al (2001) recently developed a sequential GISH and FISH approach to characterize potato germplasm containing chromosomes derived from wild species. In this approach, GISH was first used to reveal the number of chromosomes from the wild species.…”

“…The same cytological preparations were then re-probed using BAC markers specific to individual potato chromosomes. These markers are called chromosome-specific cytogenetic DNA markers (CSCDM) (Dong et al 2000(Dong et al , 2001. This sequential approach can reveal the number and identity of the alien chromosomes and their potato homologues in the breeding lines.…”

“…Fluorescence in situ hybridization (FISH) using BAC and genomic DNA probes was performed essentially as described by Dong et al (2000Dong et al ( , 2001. Bacterial artificial chromosome (BAC) clone 220C03, which was mapped to the long arm of potato chromosome 8 , was used as a chromosome-specific cytogenetic DNA marker for the identification of chromosome 8.…”

“…Chromosomes were counterstained with 4 0 ,6-diamidino-2-phenylindole (DAPI) in a Vectashield antifade solution (Vector Laboratories) and pseudocolored in red. A sequential FISH and GISH (genomic in situ hybridization) procedure (Dong et al 2001) was used to reveal the specificity of the introgressed S. brevidens chromosome in potato background. The order of the hybridizations (first FISH and then GISH) was reversed compared to our published protocols.…”