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.

Nuclear filament-containing protein 2 (Nuf2p), a centromere-associated protein, is an indispensable component of Ndc80 complex, which is important for stable microtubulekinetochore attachment, chromosome pairing, and spindle assembly in mitosis. Based upon contig sequences coding for Nuf2p from a pyrosequencing transcriptome of Saccharina japonica, primers were designed for molecular cloning of this kinetochore protein. The open reading frame (ORF) of this gene SjNuf2 was 1,452 bp long, encoding a putative protein consisting of 483 amino acids. Multi-sequence alignment showed SjNuf2p possessed several conserved domains of Nuf2, Spc7, SPT2, and MIT. To illustrate the location of SjNuf2p using immunocytochemistry, its ORF was inserted into a prokaryotic expression vector pET-28a. Then, the recombinant SjNuf2p (rSjNuf2p) was induced to be expressed in Escherichia coli. The rSjNuf2p was verified by Western blotting with the commercial anti-6 × His-tag antibody and then purified using affinity chromatography. The polyclonal antibody of anti-SjNuf2p was raised against the purified rSjNuf2p. Western blotting with this prepared antibody showed the presence of SjNuf2p in the gametophytes of a Saccharina cultivar. SjNuf2p and α/β-tubulin were subsequently co-localized to the paraffin sections of kelp chromosomes with their corresponding antibodies, thus illustrating that SjNuf2p could reside at the kelp centromeres. This study laid a solid foundation for kelp centromere and karyotype analyses.