Sequential fluorescent staining with CMA and DAPI for somatic chromosome identification in cucumber [Cucumis sativus L.]

Each of the seven chromosomes in cucumber (Cucumis sativus L.) was identified using sequential staining with Chromomycin A₃ (CMA) and 4-6-diamidino-2-phenylindole (DAPI) as DNA base-specific fluorescent dyes. The present method using enzymatic digestion produced a high level of well-spread early-metaphase chromosome complements. After CMA and DAPI staining, reproducible fluorescence bands were obtained in mitotic prometaphase chromosomes. The CMA staining method made it possible to characterize whole chromosomes from prometaphase to mid-metaphase. Chromosome 1 had the largest and widest CMA-positive (CMA⁺) band from the proximal region to the interstitial region on the long arm in prometaphase. A large gap separating of the short arm from the long arm was always observed in chromosome 2 during prometaphase. The banding pattern of the short arm was similar to that of the long arm in chromosome 2. Chromosomes 1 and 2 in early metaphase had sharp and large CMA-positive and DAPI-negative (CMA⁺DAPI⁻) bands at the pericentromeric regions. In early metaphase, chromosome 3 was characterized by having a narrow CMA⁺DAPI⁻ band on the pericentromeric region of the short arm. Chromosomes 4 and 5 showed similar chromosome length and had a large CMA⁺ band at the distal region of the long arm. Chromosome 4 did not show any clear band in the short arm, while chromosome 5 showed a telomeric CMA⁺ band at the short arm and a clear CMA⁺DAPI⁻ band at the pericentromeric region. Chromosome 6 had a CMA⁺ band at the distal region and a weak CMA⁺ band at the proximal region in each of the arms. Chromosome 7 had an evident CMA⁺ band in the long arm and a CMA⁺ DAPI⁻ band in the pericentromeric region