Application of genomic in situ hybridization to the chromosome complement of the intergeneric hybrid between Leucanthemella linearis (Matsum.) Tzuvelev and Nipponanthemum nipponicum (Franch. et Maxim.) Kitamura

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Leucanthemella linearis (2n=18) and Nipponanthemum nipponicum (2n=18) were readily crossed by hand pollination. Root tips of their F_1 hybrid were harvested, pretreated and fixed by the conventional technique before they were macerated with an enzymatic solution of 2% cellulase Onozuka RS, 1% pectolyase Y-23, 75 mM KCl and 7.5 mM EDTA at pH 4.0 for 30 min at 37℃. They were squashed with 45% acetic acid. Total genomic DNA was extracted from fresh leaves of L. linearis and N. nipponicum separately by the improved CTAB method. These DNA probes were labelled with biotin-14-dATP by the nick translation method. Combined denaturation of the genomic DNA probes and the slide preparation were conducted by an automatic temperature controller at 85℃ for 10 min and they were hybridized at 37℃ for 12-14 h. By genomic in situ hybridization, total genomic DNA probe of N. nipponicum was hybridized to nine small chromosomes of N. nipponicum yellow-FITC-fluoresced in the chromosome set at mitotic metaphase in the F_1 hybrid but not to the other nine large chromosomes of L. linearis and these were counterstained red by propidium iodide.
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