Flow karyotyping and sorting of Vicia faba chromosomes.
1993
Lucretti S. | Dolezel J. | Schubert I. | Fuchs J.
Chromosome suspensions were prepared from formaldehyde-fixed, synchronized Vicia faba root tips. After staining with the DNA intercalating fluorochrome propidium iodide, the suspensions were analysed with a flow cytometer. The resulting histograms of integral fluorescence intensity contained peaks similar to those of theoretical V. faba flow karyotypes. From V. faba cv 'Inovec' (2n=12) only 1 peak, corresponding to a single chromosome type (meta-centric chromosome), could be discriminated. However, it was found that the peak also contained doublets of acrocentric chromosomes. Bivariate analysis of fluorescence pulse area (chromosome DNA content) and fluorescence pulse width (chromosome length) was necessary to distinguish the metacentric chromosome. To achieve a high degree of purity, a 2-step sorting protocol was adopted. During a working day, more than 25 000 metacentric chromosomes (corresponding to 0.2 myg DNA) were sorted with a purity of more than 90%. Such chromosomes are suitable for physical gene mapping by in situ hybridization or via the polymerase chain reaction (PCR) and allow the construction of chromosome-specific DNA libraries. While it was only possible to distinguish and sort one chromosome type from V. faba cv 'Inovec' with the standard karyotype, it was possible to sort with a high degree of purity 5 out of 6 chromosome types of the line EFK of V. faba, which has 6 pairs of morphologically distinct chromosomes. This result confirmed the possibility of using reconstructed karyotypes to overcome existing problems with the discrimination and flow sorting of individual chromosome types in plants.
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