Characterization of repetitive DNA sequences carrying 5S rDNA of the triploid ginbuna (Japanese silver crucian carp, Carassius auratus langsdorfi).

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Abstract
Repetitive DNA sequences (Hi-b; 209 bp in length) were isolated from the HindIII digests of the genomic DNA of the triploid ginbuna, Carassius auratus langsdorfi. Sequence analyses revealed that the Hi-b repetitive units were comprised of the complete coding regions of 5S rDNA (120 bp in size) and their 5'flanking regions. The sequences of the Hi-b units from the same individual were highly homogeneous. Southern blot hybridization to the Hi-b probe displayed intricate patterns that represented the presence of other repetitive units containing the Hi-b related sequences. A major family of repetitive sequences related to the Hi-b was then obtained by the polymerase chain reaction using asymmetry primers for the 5S coding regions. These 331-bp sequences (AZ5S's) contained 5S pseudogenes as well as the almost entire Hi-b sequences, and seemed to be the true 5S rDNAs. The tandem arrangements of the AZ5S sequences explained most of the complex results of Southern blots. Another class of intriguing repeat units (Hi-b-beta and Hi-b-gamma) were also isolated. Fluorescence in situ hybridization data revealed two major signals on a pair of homologous chromosomes and several minor signals on other chromosomes in the triploid ginbuna, indicating the existence of the 5S related sequences as several separate clusters. The major spots were shared with the tetraploid ginbuna and goldfish, but not with the diploid ginbuna. When the genomic organization of the Hi-b related sequences in other cyprinid fishes was examined, the hybridization patterns of the ginbuna were very similar to those of the goldfish, but were clearly different from those of the gengorobuna. The carp genome showed less complex patterns. Thus, the present 5S rDNA-related sequences could be candidates for phylogenetic molecular markers for the crucian carp.