Abstract
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.
Highlights
The previous cytogenetic reports on the economically significant brown seaweed, Saccharina japonica (Aresch.) C
The length of the 18S ribosomal RNA (rRNA) gene was 1824 bp, and its sequence was identical to the reported 18S rDNA of the same species (GenBank Accession No EU293553, Bi et al 2009) and highly similar to those that were respectively cloned from Laminaria digitata, Saccharina angustata, Costaria costata, and Ectocarpus siliculosus (Supplementary Table 1)
The 5.8S rRNA gene of S. japonica was 160 bp in size and it was identical in size to those of C. costata (Bhattcharya and Druehl 1988) and E. siliculosus (Peters et al 2010)
Summary
The previous cytogenetic reports on the economically significant brown seaweed, Saccharina japonica (Aresch.) C. Saunders (=Laminaria japonica Aresch.), have demonstrated that kelp chromosomes are very small (0.57–2.61 μm) in size and nearly homomorphic in shape (Yabu 1973; Tai and Fang 1976, 1977; Yabu and Yasui 1991; Zhou et al 2004; Liu et al 2012a,b) These chromosomal features make it difficult to identify individual chromosomes and analyze the karyotype of brown seaweeds (Lewis 1996) by use of conventional cytogenetic techniques. Using the sensitive fluorescence in situ hybridization (FISH) technique, especially with nuclear ribosomal RNA (rRNA) genes as probes, has been proven to be efficient for the investigation of species with small or sized chromosomes (Leitch and Heslop-Harison, 1992; Garcia et al 2012). This enables the identification and discrimination of different chromosomes within and between chromosome complements (Hamon et al 2009)
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