Localization of a Female-Specific Marker on the Chromosomes of the Brown Seaweed Saccharina japonica Using Fluorescence In Situ Hybridization

Background: There is a heteromorphic alternative life in the brown seaweed, Saccharina japonica (Aresch.) C. E. Lane, C. Mayes et G. W. Saunders (=Laminaria japonica Aresch.), with macroscopic monoecious sporophytes and microscopic diecious gametophytes. Female gametophytes are genetically different from males. It is very difficult to identify the parent of a sporophyte using only routine cytological techniques due to homomorphic chromosomes. A sex-specific marker is one of the best ways to make this determination. Methodology/Principal Findings: To obtain clear images, chromosome preparation was improved using maceration enzymes and fluorochrome 4', 6-diamidino-2-phenylindole (DAPI). The chromosome number of both male and female haploid gametophytes was 31, and there were 62 chromosomes in diploid sporophytes. Although the female chromosomes ranged from 0.77 mu m to 2.61 mu m in size and were larger than the corresponding ones in the males (from 0.57 mu m to 2.16 mu m), there was not a very large X chromosome in the females. Based on the known female-related FRML-494 marker, co-electrophoresis and Southern blot profiles demonstrated that it was inheritable and specific to female gametophytes. Using modified fluorescence in situ hybridization (FISH), this marker could be localized on one unique chromosome of the female gametophytes as well as the sporophytes, whereas no hybridization signal was detected in the male gametophytes. Conclusions/Significance: Our data suggest that this marker was a female chromosome-specific DNA sequence. This is the first report of molecular marker localization on algal chromosomes. This research provides evidence for the benefit of using FISH for identifying molecular markers for sex identification, isolation of specific genes linked to this marker in the females, and sex determination of S. japonica gametophytes in the future.