Plant Genome IV Conference Plant Genome IV Conference
Chromosome sorting using flow cytometry is of great interest as it would allow construction of chromosome-specific libraries. Chromosome-specific libraries will be useful for mapping and cloning of agronomically important genes. The goal of this research was to synchronize cell cycles in maize root meristems, to develop a flow karyotype, and to construct maize chromosome-specific libraries. Effective synchronization of maize root meristematic cells was achieved by treating the seedling roots with 5 mM hydroxyurea. The highest mitotic index (over 70%) was achieved by treating the synchronized roots with 1 uM trifluralin. Suspensions of chromosomes were prepared from root tips by s simple chopping method in MgSO4 buffer and analyzed using a flow cytometer (EPICS V FACScan, FACSvantage) after staining with propidium iodide. Consistent chromosome peaks were observed. There were six peaks for ten maize chromosomes. The peaks on these experimental flow karyotypes were comparable to the peaks on a model flow karyotype of maize (constructed based on chromosome sizes). The chromosome peak corresponding to chromosome 1 was positively identified by comparing with an experimental flow karyotype obtained from a maize 1B-L translocation line. Chromosomes were sorted from each chromosome peak, and the purity was confirmed by examination of sorted fractions under a fluorescent microscope. DNA sequences specific to sorted chromosomes were amplified via DOP-PCR as well as with linker-adopter PCR methods. Putative chromosome-specific libraries are being assessed by genetic mapping and in situ hybridization.