Flow cytometric sorting of maize chromosome 9 from an oat-maize chromosome addition line

Abstract

Large numbers of maize chromosome 9 can be collected with high purity by flow cytometric sorting of chromosomes isolated from a disomic maize chromo- some addition line of oat. Metaphase chromosome sus- pensions were prepared from highly synchronized seed- ling root-tips of an oat-maize chromosome-9 addition line (OM9) and its parental oat and maize lines. Chromo- somes were stained with propidium iodide for flow cyto- metric analysis and sorting. Flow-karyotypes of the oat- maize addition line showed an extra peak not present in the parental oat line. This peak is due to the presence of a maize chromosome-9 pair within the genome of OM9. Separation of maize chromosome 9 by flow cytometric sorting of a chromosome preparation from a normal maize line was not possible because of its size similarity (DNA content) to maize chromosomes 6, 7 and 8. How- ever, it is possible to separate maize chromosome 9 from oat chromosomes and chromatids. An average of about 6× 10 3 chromosomes of maize chromosome 9 can be col- lected by flow-sorting from chromosomes isolated from 30 root tips (ten seedlings) of the oat-maize addition line. Purity of the maize chromosome 9, sorted from the oat-maize chromosome addition line, was estimated to be more than 90% based on genomic in situ hybridiza- tion analysis. Sorting of individual chromosomes pro- vides valuable genomic tools for physical mapping, li- brary construction, and gene isolation.