Characterization and physical mapping of nuclear ribosomal RNA (rRNA) genes in the haploid gametophytes of Saccharina japonica (Phaeophyta)

摘要

Nuclear tandemly repeated ribosomal RNA genes (18S-5.8S-25S rDNA and 5S rDNA) have been proven to be excellent cytogenetic markers for karyotype analysis of various higher plants by using the fluorescence in situ hybridization (FISH) technique. To illustrate physical mapping of these rDNAs in brown seaweed, Saccharina japonica, chromosomes, an approximately 5400-bp transcription unit of 18S-5.8S-25S rDNA was assembled by cloning the 25S rDNA after paired-end sequencing of two screened clones from a bacterial artificial chromosome library of kelp gametophytes. In contrast to the conserved 18S-5.8S-25S rDNA and ITS1 in S. japonica, the 245-bp ITS2 was variable in sequence. The cloned 5S rDNA revealed that the 120-bp conserved coding region was separated by a diverse intergenic spacer sequence that were 250, 445, 905, or 1335 bp in length. On average, the 18S-5.8S-25S rDNA of kelp female and male gametophytes had 45 and 41 copies per haploid genome, respectively, as detected by quantitative real-time PCR, whereas the 5S rDNA had 2590 and 2648 copies, respectively. Southern hybridization with labeled probes of 18S rDNA or 5S rDNA demonstrated that kelp gametophytes possessed only one locus each of the 18S-5.8S-25S or 5S rDNAs. This was further confirmed by FISH analysis using the same labeled probes, thus illustrating that 18S-5.8S-25S rDNA is located at the intercalary region of chromosome 23, whereas 5S rDNA at the sub-telomeric region of chromosomes 27. The localization of these rDNAs using the FISH technique has facilitated the identification of individual chromosomes and karyotype analysis of this kelp.