biologia plantarum

International journal on Plant Life established by Bohumil Němec in 1959

Biologia plantarum 51:43-48, 2007 | DOI: 10.1007/s10535-007-0009-9

Chromosome analysis and sorting in Vicia sativa using flow cytometry

P. Kovářová1, A. Navrátilová2, J. Macas2, J. Doležel1,3,*
1 Laboratory of Molecular Cytogenetics and Cytometry, Institute of Experimental Botany, Olomouc, Czech Republic
2 Institute of Plant Molecular Biology, České Budějovice, Czech Republic
3 Department of Cell Biology and Genetics, Palacký University, Olomouc, Czech Republic

Procedures were developed for flow cytometric analysis and sorting of mitotic chromosomes (flow cytogenetics) of common vetch (Vicia sativa L., 2n=12). Suspensions of intact chromosomes were prepared from root tips after cell cycle synchronization, formaldehyde fixation, and mechanical homogenization. On average, 3 × 105 morphologically intact chromosomes could be isolated from 25 root tips. Flow cytometric analysis of DAPI-stained chromosomes resulted in histograms of relative fluorescence intensity (flow karyotypes) containing four peaks, representing particular chromosomes and/or pairs of chromosomes with similar relative DNA content. Peaks I and II were assigned to chromosomes 6 and 5, respectively. These chromosomes could be sorted with a purity exceeding 90 %. The two remaining peaks on the flow karyotype were composite, each of them representing a pair of chromosomes. Chromosomes 1 and 3 were assigned to composite peak III while chromosomes 2 and 4 were assigned to composite peak IV. The chromosomes could be sorted with a purity of 99 % from both composite peaks. Bivariate flow karyotyping after simultaneous staining of chromosomes with DAPI and mithramycin was not found helpful in discriminating additional chromosomes. This study extends the number of legume species for which flow cytogenetics is available and provides a new tool for targeted and effective analysis and mapping of common vetch genome.

Keywords: cell cycle synchronization; chromosome suspension; common vetch; metaphase accumulation; physical genome mapping
Subjects: cell cycle synchronization; chromosome; common vetch; flow cytometry; hydroxyurea; mitotic index; Vicia sativa

Received: May 23, 2005; Accepted: November 25, 2005; Published: March 1, 2007  Show citation

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Kovářová, P., Navrátilová, A., Macas, J., & Doležel, J. (2007). Chromosome analysis and sorting in Vicia sativa using flow cytometry. Biologia plantarum, 51(1), 43-48. doi: 10.1007/s10535-007-0009-9
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