Wheat Chromosome Preparation and ND-FISH

Germination of wheat seeds and preparation of mitotic metaphase chromosome from root tips were followed by the description of Han et al. [48 (link)]. The protocol of non-denaturing FISH (ND-FISH) using synthesized probes was described by Fu et al. [49 (link)]. The sequence of probe Oligo-HvCSR was based on the barley-specific satellite sequence [50 (link)]. The synthetic oligonucleotides Oligo-pSc119.2, Oligo-pTa535, Oligo-Po5, Oligo-7E-716, Oligo-7E-599, Oligo-5SrDNA, Oligo-18SrDNA, Oligo-3A1, Oligo-13-J1011, Oligo-7E-744, Oligo-d01-135, Oligo-Ae334 and Oligo-(GAA)₇ were used for ND-FISH analysis and their sequences are showed in Table 1. All oligonucleotide probes were either 5′ end-labeled with 6-carboxyfluorescein (6-FAM) for green or 6-carboxytetramethylrhodamine (Tamra) for red signals. The pictures of FISH results under Olympus BX-53 microscope were taken by a DP-70 CCD camera (Olympus, Shinjuku, Japan).