Fukio Ikeda

Comparative analysis on the distribution of heterochromatin in Citrus, Poncirus and Fortunella chromosomes

Double fluorochrome staining with chromomycin A3 (CMA) and 4′-6-diamidino-2-phenylindole (DAPI) was used to characterize and compare the distribution of constitutive heterochromatin along chromosomes of Citrus, Poncirus and Fortunella species. Only CMA-positive bands were distinguishable in metaphase chromosomes. Preferential distribution of heterochromatin in terminal regions, mainly of the long arm, and centromeric regions of a few long chromosomes was a common feature of these genera. Heteromorphism between possible homologous chromosomes was present in the majority of species. Citrus and Poncirus revealed some remarkably uniform chromosomes without any intensively fluorescing region, whereas Fortunella cultivars were differentiated by the presence of CMA bands in all chromosomes. Through measurements assisted by a computer, amounts of CMA-positive regions were shown to be highest in Fortunella. Similarities between Citrus and Poncirus suggest little heterochromatin diversification among karyotypes of these genera, whereas Fortunella, with higher amounts and more homogenous distribution of heterochromatin, is more divergent.

Somatic hybrids obtained by fusion between Poncirus trifoliata (2x) and Fortunella hindsii (4x) protoplasts

Somatic hybrids were obtained by the symmetric fusion of embryogenic callus cells from tetraploid ‘Mame’ kumquat [Fortunella hindsii (Champ.) Swing.] and mesophyll cells from diploid trifoliate orange [Poncirus trifoliata (L.) Raf.]. Southern blot analysis of three regenerants revealed that they carried specific rDNA fragments from both fusion partners, thereby confirming their hybridity. In contrast, mitochondrial DNA (mtDNA) and chloroblast DNA (cpDNA) were unidirectionally transmitted from the callus parent without any evidence of recombination. No differences in the restriction fragment patterns of rDNA, mtDNA or cpDNA could be detected among the regenerants. Flow cytometry showed that two regenerants were hexaploids, as expected, but that one was pentaploid, probably due to elimination of chromosomes prior to the regeneration of this plant.

rDNA sites and heterochromatin in Meiwa kumquat (Fortunella crassifolia Swing.) chromosomes revealed by FISH and CMA/DAPI staining

SUMMARYFluorescence in situ hybridization (FISH) was used to localize rDNA sites in relation to heterochromatic regions revealed by double chromomycin A3 (CMA) and 4′-6-diamidino-2-phenylindole (DAPI) in somatic chromosomes of Meiwa kumquat (Fortunella crassifolia Swing.). Four sites were observed, all corresponding to CMA positive regions. Two of them were located in the centromeric region of the two chromosomes with three CMA bands (type A chromosome), and the other two, in the long arms terminal region of the chromosomes with terminal CMA bands on both telomeres (type C). These sites were of different sizes among probable homologous chromosomes, most likely due to a hybrid origin of this accession. The sites located in the type C chromosomes represent a new locus of rRNA genes in these taxa, suggesting that different evolutionary pathways may lead to a similar heterochromatin distribution, and, hence, that chromosomes with the same CMA banding pattern among different species may not always be homoeolo...

Chromosome markers and alterations in mitotic cells from interspecific Citrus somatic hybrids analysed by fluorochrome staining

Abstract Mitotic cells from Rough lemon (Citrus jambhiri Lush.), Ohta ponkan (C. reticulata Blanco) and two somatic hybrid plants obtained from protoplast fusion were analysed by double staining with chromomycin A3 (CMA) and 4′-6-diamidino-2-phenylindole. Only CMA-positive bands were observed in metaphasic chromosomes. The two parental karyotypes (2n=2x=18) were heteromorphic, yielding some marker chromosomes that could be identified in the somatic hybrids. One of the somatic hybrids had 2n=37 chromosomes, and the possible extra chromosome was distinguishable. The second somatic hybrid was tetraploid (2n=4x=36), with one of the chromosomes bearing a putative structural alteration. Furthermore, aneusomaty and some mitotic abnormalities were also observed in this latter plant. Such irregularities are reported for the first time for citrus somatic hybrids, and their possible causes and implications are discussed.