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

PLOS ONE, Dec 2019

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 µm to 2.61 µm in size and were larger than the corresponding ones in the males (from 0.57 µm to 2.16 µm), 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.

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

Zhou Z (2012) Localization of a Female-Specific Marker on the Chromosomes of the Brown Seaweed Saccharina japonica Using Fluorescence In Situ Hybridization. PLoS ONE 7(11): e48784. doi:10.1371/journal.pone.0048784 Localization of a Female-Specific Marker on the Chromosomes of the Brown Seaweed Saccharina japonica Using Fluorescence In Situ Hybridization Yu Liu. 0 YanHui Bi. 0 JunGang Gu 0 LiHua Li 0 ZhiGang Zhou 0 Sebastian C. A. Ferse, Leibniz Center for Tropical Marine Ecology, Germany 0 College of Aqua-life Sciences and Technology, Shanghai Ocean University , Shanghai , China 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 49, 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 mm to 2.61 mm in size and were larger than the corresponding ones in the males (from 0.57 mm to 2.16 mm), 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. - . These authors contributed equally to this work. Saccharina japonica (Aresch.) C. E. Lane, C. Mayes et G. W. Saunders ( = Laminaria japonica Aresch.) is a brown seaweed of high economic importance especially in East Asia, such as in China, Japan and South Korea, where it has been cultivated extensively for food and industrial alginate. China is by far the largest producer, and the production of L. japonica in China in 2009 rose sharply to 4.146109 kg wet weight [1], accounting for approximately 80% of the global production, over several decades. This has been attributed to both a fundamental understanding of its biology as well as the cultivation techniques and genetic breeding [25]. There are two heteromorphic alternative forms in S. japonica, macroscopic monoecious sporophytes (2n) and microscopic diecious gametophytes (n). The haploid female and male gametophytes differ from each other not only in morphology [6] and physiology [7,8], but also in genetics. The female gametophytes can develop and produce eggs and even give rise to parthenogenetic sporophytes without fertilization [912] whereas the males produce sexual spermatozoids [13]. These discrepancies possibly result from the different genes or differential expression of genes between males and females. Accordingly, attempts have been made to identify sex-specific genes by construction and characterization of subtraction cDNA libraries using suppression subtractive hybridization [14,15]. Unfortunately, no sex -specific genes were found in these studies. At the same time, a sequence-characterized amplified region (SCAR) marker, FRML-494 (GenBank accession No. EU931619), related to the female gametophytes was developed [16] on the basis of a specific sequence (GenBank accession No. AB069714) of the Marchantia polymorpha L. Y chromosome. However, no cytological studies were conducted to further characterize the sex-specific marker. In order to utilize this putative sex-specific marker as a tool for sex identification, which otherwise was possible only through morphological identification at the early developmental stage of zoospore germination, additional studies are required to provide cytological evidence of sex linkage to this putative sex-specific marker. The objective of this study was to look into the specific relationship between the marker and female gametophytes and to determine chromosomal localization using Southern blotting and fluorescence in situ hybridization (FISH). FISH was initially developed in the fi (...truncated)


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Yu Liu, YanHui Bi, JunGang Gu, LiHua Li, ZhiGang Zhou. Localization of a Female-Specific Marker on the Chromosomes of the Brown Seaweed Saccharina japonica Using Fluorescence In Situ Hybridization, PLOS ONE, 2012, 11, DOI: 10.1371/journal.pone.0048784