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Lucretti S, Doležel J, Schubert I, et al. Flow karyotyping and sorting of Vicia faba chromosomes. Theor Appl Genet. 1993;85(6):665-72doi: 10.1007/BF00225003.
Lucretti, S., Doležel, J., Schubert, I., & Fuchs, J. (1993). Flow karyotyping and sorting of Vicia faba chromosomes. TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik, 85(6), 665-72. https://doi.org/10.1007/BF00225003
Lucretti, S, et al. "Flow karyotyping and sorting of Vicia faba chromosomes." TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik vol. 85,6 (1993): 665-72. doi: https://doi.org/10.1007/BF00225003
Lucretti S, Doležel J, Schubert I, Fuchs J. Flow karyotyping and sorting of Vicia faba chromosomes. Theor Appl Genet. 1993 Feb;85(6):665-72. doi: 10.1007/BF00225003. PMID: 24196034.
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Theor Appl Genet. 1993 Feb;85(6):665-72. doi: 10.1007/BF00225003.

Flow karyotyping and sorting of Vicia faba chromosomes.

TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik

S Lucretti, J Doležel, I Schubert, J Fuchs

Affiliations

  1. Agricultural R&D Department, Genetic Engineering Division, ENEA, C.R.E. Casaccia, C.P. 2400, Rome, Italy.

PMID: 24196034 DOI: 10.1007/BF00225003

Abstract

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 one peak, corresponding to a single chromosome type (metacentric 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 two-step sorting protocol was adopted. During a working day, more than 25 000 metacentric chromosomes (corresponding to 0.2 μg 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 five out of six chromosome types of the line EFK of V. Faba, which has six 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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