Chromosome Analysis via Oligo-FISH Probes

Root tips from germinated seeds were collected and treated with nitrous oxide followed by enzyme digestion [56 (link)]. The Giemsa C-banding was done according to according to Li et al. [57 (link)]. The TR based probes with the synthetic oligos were labeled with either 5’ end-labelled 6-carboxyfluorescein (6-Fam) for green or 6-carboxytetramethylrhodamine (Tamra) for red signals. The protocol of non-denaturing FISH (ND-FISH) using synthesized probes was described by Fu et al. [58 (link)]. The wheat-barley linkage group specific bulked oligo pool probes (Synt1 to Synt 7) were designed following our recently published procedure [37 (link)]. After oligo-based FISH, sequential FISH painting with oligo pool probes was conducted according to Han et al. [38 (link)] and Bi et al. [59 (link)]. Photomicrographs were taken with an Olympus BX-53 microscope equipped with a DP-70 CCD camera.