The study of Doležel et al. (1994) indicated a great potential of flow cytometry for the analysis of the Musa genome. The applications of DNA flow cytometry involved the estimation of nuclear genome size and rapid determination of ploidy level.
During the last years, determination of ploidy level using flow cytometry was used to characterize Musa accessions stored in the ITC collection. Nowadays, determination of ploidy usinf flow cytometry is the first step of the SSR genotyping platform which was developed in our lab and is used for molecular characterization of Musa germplasm.
Ploidy analysis is performed by analyzing DNA content in nuclei isolated from leaf tissues. Briefly, suspensions of cell nuclei are prepared by mechanical homogenization of fresh leaf tissues in nuclei isolation buffer and chicken red blood cell nuclei (CRBC) were used as internal standard. DNA of isolated nuclei is stained using DNA fluorochrome DAPI and relative fluorescence intensity is measured using Partec PAS flow cytometer. Ploidy of unknown samples is determined based on the ratio of fluorescence intensity of Musa and CRBC nuclei.
Upon request, the laboratory can provide ploidy analysis of Musa accessions as a service for Musa research comunity.
Please, send your requests to: dolezel
ueb [dot] cas [dot] cz
Genome size is estimated using flow cytometry according to a protocol of Doležel et al. (1994). Briefly, cell nuclei are isolated simultaneously from ~ 50 mg of banana leaf tissues and ~ 10 mg of young leaf of soybean, which served as an internal reference standard (2C = 2.50 pg DNA). The samples are stained by propidium iodide and DNA amount in absolute units is calculated by comparing relative fluorescence intensities of G1-phase nuclei.
