Chromosome preparations from protoplasts: in situ hybridization banding pattern of a dispersed DNA sequence in rye (Secale cereale L.)

Abstract

The utilization of genome-specific DNA sequences coupled with in situ hybridization for chromosome karyotyping in wheat, rye, and triticale has been of limited value because of the presence of cellular and cytoplasmic debris. The use of protoplasts, thus eliminating cellular debris, has been shown to improve the level of detection of low-copy and unique DNA sequences in cereals. Therefore, the use of protoplasts could represent an appropriate tool to improve the results of karyotyping cereal chromosomes with genome-specific DNA sequences. This paper describes the results on the comparative application of protoplasts and squash preparations in the analysis of physical mapping of a dispersed DNA sequence (pSc119.1) to rye chromosomes by in situ hybridization. Individual chromosomes of rye were not distinguishable by their hybridization patterns to pSc119.1 when squash preparations were used. These showed an undefined distribution of the DNA probe that covered apparently the entire length of each rye chromosome. However, considerable improvement was observed for the differential distribution of the pSc119.1 DNA sequence in protoplast preparations. The karyotypic banding pattern of pSc119.1 showed a better banding pattern than can be observed using the C-banding technique. Therefore, the use of protoplasts hybridized with dispersed DNA markers could be of more value in monitoring chromosome karyotypes than existing cytological techniques.Key words: biotin labeling, dispersed sequences, rye.