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The chromosomes of trifoliate orange (Poncirus trifoliata (L.) Raf.) were stained with chromomycin A3 (CMA) and double stained with alkylating fluorochromes 4'-6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI). PI-positive (PI (+)) regions in PI/DAPI staining were identical to CMA-positive (CMA (+)) regions. The signals of PI (+) were more stable than those of CMA (+). Based on the relative-sized PI (+) signals and the morphology of no-signal chromosomes, eighteen chromosomes of trifoliate orange were classified into 9 groups. Genomic in situ hybridization (GISH) was performed on trifoliate orange chromosomes using the double probe of DNA from trifoliate orange labeled with digoxigenin-rhodamine (red), and from ‘Nankan No. 20’ satsuma mandarin (Citrus unshiu Marcow.) or ‘Tosa Buntan’ pummelo (C. maxima (Burm.) Merr.) labeled with biotinFITC (green). Sixteen GISH signals with a higher intensity were detected in the identical positions of PI (+) regions. The coloration of GISH showed 13 yellow signals and 1 green signal by the biotin probes of satsuma mandarin, and 5 yellow signals and 11 red signals by those of ‘Tosa Buntan’ pummelo. This indicates that trifoliate orange is more closely related to satsuma mandarin than ‘Tosa Buntan’ pummelo, because red signals indicate that they share no homologous sequences between genomes. GISH signals on the secondary constriction regions of all B-type chromosomes and on the telomeric region of the D4-type chromosome were yellow or green in both satsuma mandarin and ‘Tosa Buntan’ pummelo biotin probes. This result suggests that these regions in trifoliate orange chromosomes can be homologous to satsuma mandarin and ‘Tosa Buntan’ pummelo genomes, and are thus conserved regions.

Chromosome Identification and Characterization in Trifoliate Orange (Poncirus trifoliata (L.) Raf.) by CMA and PI/DAPI Staining and GISH