The physical localization of the 6 tandem repeats (TRs) was analyzed by the FISH procedure. A chromosome spread preparation was made from root tip as described in [28 (link)]. Good slides with well-spread chromosomes were stored at 4 °C until use. FISH was carried out following the procedure in [60 (link),61 (link)]. After hybridization, the chromosomes were counterstained with 1 mg/mL DAPI. The detection was performed using streptavidin-conjugated Cy3 or FITC (Roche, Basel, Switzerland); signals in all variants were visualized using an AxioZeiss Imager V1 fluorescence microscope.
After post-hybridization washing of WWGH chromosome mitotic preparations as described in Komuro et al. [62 (link)] multicolor genomic in situ hybridization (mcGISH) was conducted on the same slides as described in Kishii et al. [63 (link)] with modifications described in Salina et al. [15 (link)]. The probes were the Pseudoroegneria spicata (St genome) and Dasypyrum villosum (V genome) genomic DNA (50 ng/preparation) labeled with digoxigenin-11-dUTP and with biotin-16-dUTP, respectively, by nick translation, according to the manufacturer’s instructions (Roche, Germany). The chromosomes were counterstained with 1 mg/mL DAPI and mounted in Vectashild (Vector Laboratories, Peterborough, UK).
The results were recorded with an AxioCam Mrm Zeiss camera and contrasted using AxioVision software.
After post-hybridization washing of WWGH chromosome mitotic preparations as described in Komuro et al. [62 (link)] multicolor genomic in situ hybridization (mcGISH) was conducted on the same slides as described in Kishii et al. [63 (link)] with modifications described in Salina et al. [15 (link)]. The probes were the Pseudoroegneria spicata (St genome) and Dasypyrum villosum (V genome) genomic DNA (50 ng/preparation) labeled with digoxigenin-11-dUTP and with biotin-16-dUTP, respectively, by nick translation, according to the manufacturer’s instructions (Roche, Germany). The chromosomes were counterstained with 1 mg/mL DAPI and mounted in Vectashild (Vector Laboratories, Peterborough, UK).
The results were recorded with an AxioCam Mrm Zeiss camera and contrasted using AxioVision software.
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