Flow Karyotyping of Wheat Addition Line “T240” with a Haynaldia villosa 6VS Telosome
To construct a chromosome-specific DNA library of chromosome 6VS from Haynaldia villosa, a wheat alien chromosome addition line T240 with a 6VS chromosome arm and its parental common wheat cv. CA921 were used to optimize protocols for preparing chromosome suspension amenable to flow sorting of the 6VS chromosome. Our results showed that root tips incubated sequentially in Hogland’s solution containing 1.25 mM hydroxyurea (DNA synthesis inhibitor) for 18 h, a hydroxyurea-free period for 2 h, and 1 μM trifluralin for 4 h (metaphase blocking reagent) increased the metaphase index (MI) by up to 62 % . Many metaphase chromosomes were released to form a chromosome suspension when root tips fixed in 2 % paraformaldehyde were treated in a homogenizer at 9,500 rpm for 10–15 s. Most of the released chromosomes had intact morphology. The background solution of chromosome suspension was clear and relatively few of cell debris and chromosome clumps. Univariate flow karyotypes were established with chromosome suspension flow through FACS Vantage 2000 SE flow cytometer. The flow karyotype of CA921 consisted of four chromosome peaks, whereas that for T240 had a fifth peak. This fifth peak in T240 contained the telosome, which was further confirmed to be 6VS through fluorescence in situ hybridization.
KeywordsFlow cytometry Chromosome sorting FISH
We sincerely thank the Nature Science Foundation of Shaanxi province of China for support.
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